UVA causes specific mutagenic DNA damage through ROS production, rather than CPD formation, in Drosophila larvae.

Evidence is accumulating that ultraviolet A (UVA) plays an important role in photo-carcinogenesis. However, the types of DNA damage involved in the resulting mutations remain unclear. Previously, using Drosophila, we found that UVA from light-emitting diode (LED-UVA) induces double-strand breaks in DNA through oxidative damage in an oxidative damage-sensitive (urate-null) strain. Recently, it was proposed that cyclobutane pyrimidine dimers (CPDs), which also are induced by UVA irradiation, might play a significant role in the induction of mutations. In the present study, we investigated whether reactive oxygen species (ROS) and CPDs are produced in larval bodies following LED-UVA irradiation. In addition, we assessed the somatic cell mutation rate in urate-null Drosophila induced by monochromatic UVA irradiation. The production of ROS through LED-UVA irradiation was markedly higher in the urate-null strain than in the wild-type Drosophila. CPDs were detected in the DNA of both of UVA- and UVB-irradiated larvae. The level of CPDs was unexpectedly higher in the wild-type strain than in urate-null flies following UVA irradiation, whereas this parameter was expectedly similar between the urate-null and wild-type Drosophila following UVB irradiation. The somatic cell mutation rate induced by UVA irradiation was higher in the urate-null strain than in the wild-type strain. These results suggest that mutations induced by UVA-specific pathways occur through ROS production, rather than via CPD formation.

Mutation and apoptosis are well-coordinated for protecting against DNA damage-inducing toxicity in Drosophila.

BACKGROUND: Apoptotic cell death is an important survival system for multicellular organisms because it removes damaged cells. Mutation is also a survival method for dealing with damaged cells in multicellular and also unicellular organisms, when DNA lesions are not removed. However, to the best of our knowledge, no reports have comprehensively explored the direct relationship between apoptosis and somatic cell mutations induced by various mutagenic factors. RESULTS: Mutation was examined by the wing-spot test, which is used to detect somatic cell mutations, including chromosomal recombination. Apoptosis was observed in the wing discs by acridine orange staining in situ. After treatment with chemical mutagens, ultraviolet light (UV), and X-ray, both the apoptotic frequency and mutagenic activity increased in a dose-dependent manner at non-toxic doses. When we used DNA repair-deficient Drosophila strains, the correlation coefficient of the relationship between apoptosis and mutagenicity, differed from that of the wild-type. To explore how apoptosis affects the behavior of mutated cells, we determined the spot size, i.e., the number of mutated cells in a spot. In parallel with an increase in apoptosis, the spot size increased with MNU or X-ray treatment dose-dependently; however, this increase was not seen with UV irradiation. In addition, BrdU incorporation, an indicator of cell proliferation, in the wing discs was suppressed at 6 h, with peak at 12 h post-treatment with X-ray, and that it started to increase again at 24 h; however, this was not seen with UV irradiation. CONCLUSION: Damage-induced apoptosis and mutation might be coordinated with each other, and the frequency of apoptosis and mutagenicity are balanced depending on the type of DNA damage. From the data of the spot size and BrdU incorporation, it is possible that mutated cells replace apoptotic cells due to their high frequency of cell division, resulting in enlargement of the spot size after MNU or X-ray treatment. We consider that the induction of mutation, apoptosis, and/or cell growth varies in multi-cellular organisms depending on the type of the mutagens, and that their balance and coordination have an important function to counter DNA damage for the survival of the organism.

Mismatch repair systems might facilitate the chromosomal recombination induced by N-nitrosodimethylamine, but not by N-nitrosodiethylamine, in Drosophila.

Mismatch repair (MMR) systems play important roles in maintaining the high fidelity of genomic DNA. It is well documented that a lack of MMR increases the mutation rate, including base exchanges and small insertion/deletion loops; however, it is unknown whether MMR deficiency affects the frequency of chromosomal recombination in somatic cells. To investigate the effects of MMR on chromosomal recombination, we used the Drosophila wing-spot test, which efficiently detects chromosomal recombination. We prepared MMR (MutS)-deficient flies (spel1(-/-)) using a fly line generated in this study. The spontaneous mutation rate as measured by the wing-spot test was slightly higher in MutS-deficient flies than in wild-type (spel1(+/-)) flies. Previously, we showed that N-nitrosodimethylamine (NDMA)-induced chromosomal recombination more frequently than N-nitrosodiethylamine (NDEA) in Drosophila. When the wing-spot test was performed using MMR-deficient flies, unexpectedly, the rate of NDMA-induced mutation was significantly lower in spel1(-/-) flies than in spel1(+/-) flies. In contrast, the rate of mutation induced by NDEA was higher in spel1(-/-) flies than in spel1(+/-) flies. These results suggest that in Drosophila, the MutS homologue protein recognises methylated DNA lesions more efficiently than ethylated ones, and that MMR might facilitate mutational chromosomal recombination due to DNA double-strand breaks via the futile cycle induced by MutS recognition of methylated lesions.

Chloroethylating anticancer drug-induced mutagenesis and its repair in Escherichia coli.

BACKGROUND: Chloroethylnitrosourea (CENU) derivatives, such as nimustine (ACNU) and carmustine (BCNU), are employed in brain tumor chemotherapy due to their ability to cross the blood-brain barrier. They are thought to suppress tumor development through DNA chloroethylation, followed by the formation of interstrand cross-links (ICLs) that efficiently block replication and transcription. However, the alkylation of DNA and ICLs may trigger genotoxicity, leading to tumor formation as a side effect of the chemotherapeutic treatment. Although the involvement of O 6-alkylguanine-DNA alkyltransferase (AGT) in repairing chloroethylated guanine (O 6-chloroethylguanine) has been reported, the exact lesion responsible for the genotoxicity and the pathway responsible for repairing it remains unclear. RESULTS: We examined the mutations induced by ACNU and BCNU using a series of Escherichia coli strains, CC101 to CC111, in which reverse mutations due to each episome from F'101 to F'106 and frameshift mutations due to each episome from F'107 to F'111 could be detected. The mutant frequency increased in E. coli CC102, which can detect a GC to AT mutation. To determine the pathway responsible for repairing the CENU-induced lesions, we compared the frequency of mutations induced by CENU in the wild-type strain to those in the ada, ogt (AGT-deficient) strain, uvrA (nucleotide excision repair (NER)-deficient) strain, mismatch repair (MMR)-deficient strains, and recA (recombination deficient) strain of E. coli CC102. The frequencies of mutations induced by ACNU and BCNU increased in the ada, ogt strain, demonstrating that O 6-chloroethylguanines were formed, and that a portion was repaired by AGT.Mutation induced by ACNU in NER-deficient strain showed a similar profile to that in AGT-deficient strain, suggesting that an NER and AGT play at the similar efficacy to protect E. coli from mutation induced by ACNU. O 6-Chloroethylguanine is reported to form ICLs if it is not repaired. We examined the survival rates and the frequencies of mutations induced by ACNU and BCNU in the uvrA strain, the recA strain, as well as a double-deficient strain of CC102. The mutation profile of the double-deficient strain was similar to that of the NER-deficient strain, suggesting that an NER protects E. coli from mutations but not recombination. In addition, cell death was more pronounced in the uvrA, recA double-deficient strain than in the single-deficient strains. CONCLUSION: These results suggest that the toxic lesions induced by CENU were repaired additively or synergistically by NER and recombination. In other words, lesions, such as ICLs, appear to be repaired by NER and recombination independently.

2,6-Dimethoxy-1,4-benzoquinone, isolation and identification of anti-carcinogenic, anti-mutagenic and anti-inflammatory component from the juice of Vitis coignetiae.

Previously we demonstrated the anti-tumorigenic, anti-mutagenic and anti-inflammatory effects of the juice of Vitis coignetiae (yamabudo), and identified caftaric acid as an anti-mutagenic component from the juice. In the present study, we investigated the isolation of anti-inflammatory components in yamabudo juice supposing that the anti-inflammatory components in yamabudo are also responsible for the anti-tumorigenic activity. The suppressing effect on nitric oxide production in mouse leukemic monocyte with LPS was used as a separation marker. Three components comprising 2,6-dimethoxy-1,4-benzoquinone (DBQ), fertaric acid and caftaric acid were isolated and identified from the juice of V. coignetiae as anti-inflammatory ingredients. Inhibitory effects were found of DBQ on the mutagenicity of dimethylbenzo[a]anthracene, aflatoxin B1, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the Ames test. Topical application of DBA significantly inhibited TPA-induced edema of mouse ears. The anti-tumorigenic effect of DBQ on the promotion and initiation stages of mouse skin tumorigenesis was investigated, and topical administration of DBQ on the promotion stage significantly decreased tumor development in mice skin. DBQ is a potential candidate for the chemopreventive effect of V. coignetiae.

Increase of somatic cell mutations in oxidative damage-sensitive drosophila.

BACKGROUND: Oxidative damage is an important genotoxic source for almost all organisms. To efficiently detect mutations induced by oxidative damage, we previously developed a urate-null Drosophila strain. Using this Drosophila strain, we showed the mutagenic activity of environmental cigarette smoke (ECS) and the herbicide paraquat, which are known to produce reactive oxygen species (ROS). In the present study, we examined the mutagenic activities of carcinogenic mutagens that are considered to cause mutations by adduct formation, alkylation, or crosslinking of cellular DNA in the oxidative damage-sensitive Drosophila to evaluate how the oxidative damage induced by these mutagens is involved in causing mutations. In addition, we evaluated whether these oxidative damage-sensitive flies may be useful for mutation assays. METHODS: We performed the wing-spot test in oxidative damage-sensitive Drosophila (urate-null strains) to examine the mutagenicity of 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx), mitomycin C (MMC), 4-nitroquinoline N-oxide (4NQO), N-nitrosodimethyl-amine (NDMA), and N-nitrosodiethylamine (NDEA). We also observed the mutagenicity of X-ray irradiation as a control in which mutations should be mainly caused by oxidative damage. RESULTS: As expected, the mutagenic activity of X-ray irradiation was higher in the urate-null Drosophila than in the wild-type Drosophila. The mutagenic activities of the tested compounds were also higher in the urate-null Drosophila than in the wild-type Drosophila. In experiments using another urate-null strain, the mutagenicity of N-nitrosodialkylamines was also higher in the urate-null flies than in the wild-type ones. CONCLUSIONS: The tested compounds in this study were more mutagenic in urate-null Drosophila than in wild-type Drosophila. It was supposed that ROS were generated and that the ROS might be involved in mutagenesis. The present results support the notion that in addition to causing DNA lesions via adduct formation, alkylation, or DNA crosslinking, these mutagens also cause mutations via ROS-induced DNA damage. As such, urate-null Drosophila appear to be useful for detecting the mutagenic activity of various mutagens, especially those that produce reactive oxygen. If the mutation rate increases on a mutation assay using urate-null Drosophila, it might suggest that the mutagen generates ROS, and that the produced ROS is involved in causing mutations.

E. coli mismatch repair enhances AT-to-GC mutagenesis caused by alkylating agents.

Alkylating agents are known to induce the formation of O6-alkylguanine (O6-alkG) and O4-alkylthymine (O4-alkT) in DNA. These lesions have been widely investigated as major sources of mutations. We previously showed that mismatch repair (MMR) facilitates the suppression of GC-to-AT mutations caused by O6-methylguanine more efficiently than the suppression of GC-to-AT mutations caused by O6-ethylguanine. However, the manner by which O4-alkyT lesions are repaired remains unclear. In the present study, we investigated the repair pathway involved in the repair of O4-alkT. The E. coli CC106 strain, which harbors Δprolac in its genomic DNA and carries the F'CC106 episome, can be used to detect AT-to-GC reverse-mutation of the gene encoding ß-galactosidase. Such AT-to-GC mutations should be induced through the formation of O4-alkT at AT base pairs. As expected, an O6-alkylguanine-DNA alkyltransferase (AGT) -deficient CC106 strain, which is defective in both ada and agt genes, exhibited elevated mutant frequencies in the presence of methylating agents and ethylating agents. However, in the UvrA-deficient strain, the methylating agents were less mutagenic than in wild-type, while ethylating agents were more mutagenic than in wild-type, as observed with agents that induce O6-alkylguanine modifications. Unexpectedly, the mutant frequencies decreased in a MutS-deficient strain, and a similar tendency was observed in MutL- or MutH-deficient strains. Thus, MMR appears to promote mutation at AT base pairs. Similar results were obtained in experiments employing double-mutant strains harboring defects in both MMR and AGT, or MMR and NER. E. coli MMR enhances AT-to-GC mutagenesis, such as that caused by O4-alkylthymine. We hypothesize that the MutS protein recognizes the O4-alkT:A base pair more efficiently than O4-alkT:G. Such a distinction would result in misincorporation of G at the O4-alkT site, followed by higher mutation frequencies in wild-type cells, which have MutS protein, compared to MMR-deficient strains.

Inhibitory effect of Actinidia arguta on mutagenesis, inflammation and two-stage mouse skin tumorigenesis.

BACKGROUND: Actinidia arguta, known as sarunashi in Japan, is a vine tree native to east-Asia, including Japan, that produces small fruit rich in anthocyanins, catechins, vitamin C, chlorophyll, beta-carotene and other polyphenols. RESULTS: Our study revealed the inhibitory effect of the juice of A. arguta (arguta-juice) toward the mutagenicity of food-derived carcinogens and polycyclic aromatic hydrocarbons using the Ames test, and antioxidant activity of arguta-juice as determined using a free radical scavenging assay. The formation of DNA adducts in liver of mice fed 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) decreased significantly following administration of arguta-juice. The preventive effect of arguta-juice on the induction of inflammation of mouse ear by 12-O-tetradecanoylphorbol-13-acetate (TPA) was revealed. The anti-carcinogenic effect of a topically applied partially purified fraction of A. arguta was revealed on skin tumorigenesis in mice induced by treatment with 7,12-dimethylbenz(a)anthracene and TPA. In an effort to reveal the mechanisms for antimutagenicity of arguta-juice, effects on the enzymes that metabolize xenobiotics were examined. Combined effects comprising i) inhibition of the metabolic activation of mutagens with phase I enzymes, but ii) no prevention on the activity of phase II detoxification enzyme, UGT, were observed. We also investigated the characterization and partial purification of the antimutagenic components in A. arguta, which suggested that the components in A. arguta responsible for the antimutagenicity were water-soluble, heat-labile phenolic compounds. CONCLUSIONS: These results suggested that components in A. arguta are attractive candidates for potential use as chemopreventive agents.

Indolo[3,2-b]quinoline Derivatives Suppressed the Hemolytic Activity of Beta-Pore Forming Toxins, Aerolysin-Like Hemolysin Produced by Aeromonas sobria and Alpha-Hemolysin Produced by Staphylococcus aureus.

In an attempt to discover inhibitory compounds against pore-forming toxins, some of the major toxins produced by bacteria, we herein examined the effects of four kinds of indolo[3,2-b]quinoline derivatives on hemolysis induced by the aerolysin-like hemolysin (ALH) of Aeromonas sobria and also by the alpha-hemolysin of Staphylococcus aureus. The results showed that hemolysis induced by ALH was significantly reduced by every derivative, while that induced by alpha-hemolysis was significantly reduced by three out of the four derivatives. However, the degrees of reduction induced by these derivatives were not uniform. Each derivative exhibited its own activity to inhibit the respective hemolysin. Compounds 1 and 2, which possessed the amino group bonding the naphthalene moiety at the C-11 position of indolo[3,2-b]quinoline, had strong inhibitory effects on the activity of ALH. Compound 4 which consisted of benzofuran and quinoline had strong inhibitory effects on the activity of alpha-hemolysin. These results indicated that the amino group bonding the naphthalene moiety of compounds 1 and 2 assisted in their ability to inhibit ALH activity, while the oxygen atom at the 10 position of compound 4 strengthened its interaction with alpha-hemolysin. These compounds also suppressed the hemolytic activity of the supernatant of A. sobria or A. hydrophila, suggesting that these compounds were effective at the site of infection of these bacteria.

Somatic-cell mutation induced by short exposures to cigarette smoke in urate-null, oxidative stress-sensitive Drosophila.

We previously reported that a urate-null strain of Drosophila is hypersensitive to cigarette smoke (CS), and we suggested that CS induces oxidative stress in Drosophila because uric acid is a potent antioxidant. Although the carcinogenic risk of CS exposure is widely recognized; documentation of in vivo genotoxic activity of environmental CS, especially gaseous-phase CS, remains inconclusive. To date, somatic-cell mutations in Drosophila resulting from exposure to CS have not been detected via the somatic mutation and recombination test (wing spot test) with wild-type flies, a widely used Drosophila assay for the detection of somatic-cell mutation; moreover, genotoxicity has not been documented via a DNA repair test that involves DNA repair-deficient Drosophila. In this study, we used a new Drosophila strain (y v ma-l; mwh) to examine the mutagenicity induced by gaseous-phase CS; these flies are urate-null due to a mutation in ma-l, and they are heterozygous for multiple wing hair (mwh), a mutation that functions as a marker for somatic-cell mutation. In an assay with this newly developed strain, a superoxide anion-producing weed-killer, paraquat, exhibited significant mutagenicity; in contrast, paraquat was hardly mutagenic with a wild-type strain. Drosophila larvae were exposed to CS for 2, 4 or 6h, and then kept at 25°C on instant medium until adulthood. After eclosion, mutant spots, which consisted of mutant hairs on wings, were scored. The number of mutant spots increased significantly in an exposure time-dependent manner in the urate-null females (ma-l (-/-)), but not in the urate-positive females (ma-l (+/-)). In this study, we showed that short-term exposure to CS was mutagenic in this in vivo system. In addition, we obtained suggestive data regarding reactive oxygen species production in larva after CS exposure using the fluorescence probe H2DCFDA. These results suggest that oxidative damage, which might be countered by uric acid, was partly responsible for induction of somatic cell mutations in Drosophila larvae exposed to CS.

Mutagenicity and clastogenicity of extracts of Helicobacter pylori detected by the Ames test and in the micronucleus test using human lymphoblastoid cells.

Epidemiological studies have demonstrated a close association between infection with Helicobacter pylori (H.pylori) and the development of gastric carcinoma. Chronic H.pylori infection increases the frequency of mutation in gastric epithelial cells. However, the mechanism by which infection of H.pylori leads to mutation in gastric epithelial cells is unclear. We suspected that components in H.pylori may be related to the mutagenic response associated with DNA alkylation, and could be detected with the Ames test using a more sensitive strain for alkylating agents. Our investigation revealed that an extract of H.pylori was mutagenic in the Ames test with Salmonella typhimurium YG7108, which is deficient in the DNA repair of O(6)-methylguanine. The extract of H.pylori may contain methylating or alkylating agents, which might induce O (6)-alkylguanine in DNA. Mutagenicity of the alkylating agents N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine in the Ames test with S.typhimurium TA1535 was enhanced significantly in the presence of the extract of H.pylori. The tested extracts of H.pylori resulted in a significant induction of micronuclei in human-derived lymphoblastoid cells. Heat instability and dialysis resistance of the extracts of H.pylori suggest that the mutagenic component in the extracts of H.pylori is a heat-unstable large molecule or a heat-labile small molecule strongly attached or adsorbed to a large molecule. Proteins in the extracts of H.pylori were subsequently fractionated using ammonium sulphate precipitation. However, all fractions expressed enhancing effects toward MNU mutagenicity. These results suggest the mutagenic component is a small molecule that is absorbed into proteins in the extract of H.pylori, which resist dialysis. Continuous and chronic exposure of gastric epithelial cells to the alkylative mutagenic component from H.pylori chronically infected in the stomach might be a causal factor in the gastric carcinogenesis associated with H.pylori.

Somatic cell mutations caused by 365 nm LED-UVA due to DNA double-strand breaks through oxidative damage.

Evidence is accumulating indicating that UVA (320-400 nm ultraviolet light) plays an important role in photo-carcinogenesis. UVA is thought to produce reactive oxygen species in irradiated cells through photo-activation of inherent photosensitizers, and was recently reported to cause DNA double-strand breaks (DSBs) in exposed cells. We have investigated the involvement of UVA in mutations and DNA damage in somatic cells using Drosophila melanogaster larvae. Using the Okazaki Large Spectrograph, we previously observed that longer wavelength UVA (>330 nm) was more mutagenic in post-replication repair-deficient D. melanogaster (mei-41) than in the nucleotide excision repair-deficient strain (mei-9). LED-light has recently been developed as a high-dose-rate UVA source. LED-UVA light (365 nm) was also more mutagenic in mei-41 than in mei-9. The mei-41 gene was shown to be an orthologue of the human ATR gene, which is involved in the repair of DSBs through phosphorylation of histone H2AX. In order to estimate the extent to which oxidative damage contributes to mutation, we established a new D. melanogaster strain (urate-null mutant) that is sensitive to oxidative damage and has a marker to detect somatic cell mutations. When somatic cell mutations were examined using this strain, LED-UVA was mutagenic in the urate-null strain at doses that were non-mutagenic in the urate-positive strain. In an effort to investigate the generation of DSBs, we examined the presence of phosphorylated histone H2AvD (H2AX D. melanogaster homologue). At high doses of LED-UVA (>800 kJ m(-2)), levels of phosphorylated H2AvD (γ-H2AvD) increased significantly in the urate-null strain. Moreover, the level of γ-H2AvD increased in the excision repair-deficient strain but not in the ATR-deficient strain following UVA-irradiation. These results supported the notion that the generation of γ-H2AvD was mediated by the function of the mei-41 gene. It was reported that ATR functions on DSB repair in D. melanogaster. Taken together, we propose a possible pathway for UVA-induced mutation, whereby DNA double-strand breaks resulting from oxidative stress might be responsible for UVA-induced mutation in somatic cells of D. melanogaster larvae.

Properties of hemolysin and protease produced by Aeromonas trota.

We examined the properties of exotoxins produced by Aeromonas trota (A. enteropelogenes), one of the diarrheagenic species of Aeromonadaceae. Nine of 19 A. trota isolates that grew on solid media containing erythrocytes showed hemolytic activity. However, the hemolytic activities of the culture supernatants of these hemolytic strains of A. trota were markedly lower than those of A. sobria when cultured in liquid medium, and the amount of hemolysin detected by immunoblotting using antiserum against the hemolysin produced by A. sobria was also low. A mouse intestine loop assay using living bacterial cells showed that A. trota 701 caused the significant accumulation of fluid, and antiserum against the hemolysin produced suppressed the enterotoxic action of A. trota 701. These results indicated that A. trota 701 was diarrheagenic and the hemolysin produced was the causative agent of the enterotoxic activity of A. trota. The hemolysin in A. sobria was previously shown to be secreted in a preform (inactive form) and be activated when the carboxy-terminal domain was cleaved off by proteases in the culture supernatant. Since mature hemolysin was detected in the culture supernatants of A. trota, we analyzed the extracellular protease produced by A. trota. Fifteen of 19 A. trota isolates that grew on solid media containing skim milk showed proteolytic activity. We subsequently found that most A. trota isolates possessed the serine protease gene, but not the metalloprotease gene. Therefore, we determined the nucleotide sequence of the serine protease gene and its chaperone A. trota gene. The results obtained revealed that the deduced amino acid sequences of serine protease and the chaperone were homologous to those of A. sobria with identities of 83.0% and 75.8%, respectively.

Spermidine-analogous triamines suppressed the growth of Candida albicans.

We examined the antifungal activity of various synthetic triamines on several fungi. Among various triamines having a general structure H2N(CH2)aNH(CH2)bNH2 (a=2-5, b=3-8), some triamines (a=4 or 5) showed inhibitory effect on the growth of Candida albicans and C. tropicalis. Determination of the minimum inhibitory concentrations (MICs) of these triamines on C. albicans showed that triamine 4-8 (a=4, b=8) and triamine 5-8 had strong antifungal activity. Further analysis revealed that the antifungal effect of triamine 4-8 was fungistatic and the antifungal effect was diminished by the addition of spermidine, a physiological triamine, to the medium. These results suggested that triamine 4-8 is antagonistic to spermidine and the antifungal activity is due to the suppression of the action of intrinsic polyamines. On the agar medium, C. albicans formed microcolonies even in the presence of triamine 4-8 by long cultivation. We then observed the form of C. albicans using microscope and found that the cells cultivated with triamine 4-8 were round, similar to the yeast form, while most of the cells on the agar medium without triamine 4-8 were hyphal form. Subsequently, we investigated the synergistic effect of two compounds with triamine 4-8, cyclohexylamine and dl-α-difluoromethylornithine which are inhibitors of enzymes involving in the biosynthesis of physiological polyamines such as spermidine. The results showed that the antifungal activity of triamine 4-8 increased by the addition of these enzyme inhibitors.

Analysis of carboxy terminal domain of metalloprotease of elastolytic Aeromonas hydrophila.

We examined the ability of Aeromonas hydrophila to lyse elastin. Eight of 13 strains showed elastolytic activity on agar medium containing elastin and 5 strains did not. In order to examine the involvement of the metalloprotease of A. hydrophila (AMP) in elastolytic activity, we made the amp-deletion mutant strain from an elastolytic strain. The elastolytic activity of the strain decreased with this deletion. The analysis of AMP released into the culture supernatant showed that AMP appeared outside of the cell as the intermediate consisting of a mature domain and carboxy terminal (C-terminal) propeptide domain. Further analysis showed that the intermediate has the ability to lyse elastin and that loss of the C-terminal domain causes loss of the elastolytic activity of the intermediate. We then determined the nucleotide sequence of the amps of all strains used in this study. Phylogenetic analysis revealed that these AMPs were divided into three groups. The AMPs from elastolytic strains belong to group I or group II, and AMPs from non-elastolytic strains belong to group III. The distance between group I and group II is small, but group III is located separately from groups I and II. Comparison of the amino acid residues of the C-terminal domain revealed that there are 13 amino acid residues specific to the C-terminal domain of group III. This indicates that the conformation of the C-terminal propeptide domain formed by these specific amino acid residues is important for AMP to express elastolytic activity.

Distinct pathways for repairing mutagenic lesions induced by methylating and ethylating agents.

DNA alkylation damage can be repaired by nucleotide excision repair (NER), base excision repair (BER) or by direct removal of alkyl groups from modified bases by O(6)-alkylguanine DNA alkyltransferase (AGT; E.C. 2.1.1.63). DNA mismatch repair (MMR) is also likely involved in this repair. We have investigated alkylation-induced mutagenesis in a series of NER- or AGT-deficient Escherichia coli strains, alone or in combination with defects in the MutS, MutL or MutH components of MMR. All strains used contained the F'prolac from strain CC102 (F'CC102) episome capable of detecting specifically lac GC to AT reverse mutations resulting from O(6)-alkylguanine. The results showed the repair of O(6)-methylguanine to be performed by AGT ≫ MMR > NER in order of importance, whereas the repair of O(6)-ethylguanine followed the order NER > AGT > MMR. Studies with double mutants showed that in the absence of AGT or NER repair pathways, the lack of MutS protein generally increased mutant frequencies for both methylating and ethylating agents, suggesting a repair or mutation avoidance role for this protein. However, lack of MutL or MutH protein did not increase alkylation-induced mutagenesis under these conditions and, in fact, reduced mutagenesis by the N-alkyl-N-nitrosoureas MNU and ENU. The combined results suggest that little or no alkylation damage is actually corrected by the mutHLS MMR system; instead, an as yet unspecified interaction of MutS protein with alkylated DNA may promote the involvement of a repair system other than MMR to avoid a mutagenic outcome. Furthermore, both mutagenic and antimutagenic effects of MMR were detected, revealing a dual function of the MMR system in alkylation-exposed cells.

Chemopreventive effects of the juice of Vitis coignetiae Pulliat on two-stage mouse skin carcinogenesis.

Our study revealed the inhibitory effect of Vitis coignetiae Pulliat, known as Yamabudo in Japan, at the stages of multi-step carcinogenesis. The juice of Vitis coignetiae (Y-grape juice) was antimutagenic toward dimethylbenzo[a]anthracene (DMBA), aflatoxin B1, and benzo[a]pyrene in the Ames test. The Y-grape juice was also antigenotoxic in the micronucleus test using HepG2 cells toward DMBA and aflatoxin B1. Topical and oral administration of the Y-grape juice to mice inhibited the induction of inflammation of 12-O-tetradecanoylphorbol-13-acetate (TPA). Topical and oral administration of the Y-grape juice significantly decreased the incidence and mean number of tumors in mice skin with the 2-stage tumorigenesis protocol. To elucidate the mechanisms underlying the antiinflammatory and antitumor promotion activity of the Y-grape juice, the effect of Y-grape juice on cyclooxygenase-2 (COX-2) activity in mouse ear treated with TPA was studied. Both topical and oral application of the Y-grape juice inhibited the TPA-induced increase in COX-2 activity. Caftaric acid, isolated and identified from the Y-grape juice, was antimutagenic toward DMBA and prevented TPA-induced inflammation in mice, suggesting caftaric acid participates in chemopreventive effect/activities of Y-grape juice.

Effect of aflatoxin B1 on UDP-glucuronosyltransferase mRNA expression in HepG2 cells.

Aflatoxin B1 (AFB1) is a potent mycotoxin that induces hepatocellular carcinoma in many animal species, including humans. In this study, we examined the effects of AFB1 on UDP-glucuronosyltransferase (UGT) mRNA expression in HepG2 cells (human hepatocellular carcinoma cell line). The cells were treated with AFB1 for 48 h at a concentration of 10 µM, and their viability (87%) was not significantly different from that of control cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated that the mRNAs of four UGT1As (UGT1A1, UGT1A3, UGT1A4 and UGT1A9) and seven UGT2Bs (UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17 and UGT2B28) are expressed in HepG2 cells. The mRNAs of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), retinoid X receptor (RXR) and glucocorticoid receptor (GR) as transcriptional regulators were also detected. AFB1 significantly increased mRNA levels of UGT1A3, UGT2B10, UGT2B15 and UGT2B17 in HepG2 cells to 2.5-, 2.0-, 1.9- and 1.5-fold, respectively, whereas the mRNA levels of transcriptional regulators were hardly affected by AFB1. These findings suggest that AFB1 induces UGT2B isoforms rather than UGT1A isoforms in HepG2 cells, and that the change may closely contribute to the toxicity of AFB1.

Production and properties of lipase of Aeromonas sobria.

Aeromonas have been isolated from a wide variety of aquatic environments. However the number of Aeromonas in sea water is extremely small compared to that in fresh water. In in vitro culture, Aeromonas can grow in mediums containing NaCl at a concentration of 3.0%, this concentration corresponding to that of sea water. It is unclear why the number of Aeromonas is low in sea water. Exoproteins of bacteria are thought to be important for bacterial growth and survival in the environment. Previously, the present authors have shown that mediums containing 3.0% NaCl suppress production of two proteases, serine protease and metalloprotease. In this experiment, other exoproteins whose production is influenced by the amount of NaCl in the medium were analyzed. A protein whose production is repressed in medium containing 3.0% NaCl was found and purified. Biological assay of the purified protein showed that it degrades tributyrin and hydrolyzes para-nitrophenyl-fatty acylesters. These results show that the protein is a lipase. Subsequently, the nucleotide sequence of the gene encoding the lipase was determined and the amount of mRNA of the lipase gene in the cells measured. It was found that transcription of the gene is not inhibited by NaCl in the medium. This result indicates that the lipase might be synthesized, but the folding process to become an active structure does not progress smoothly in a medium containing 3.0% NaCl.

Inhibition of biosynthesis of metalloprotease of Aeromonas sobria by sodium chloride in the medium.

The present authors have previously shown that the serine protease activity of Aeromonas sobria is markedly decreased when A. sobria is cultured in medium containing 3.0% sodium chloride (NaCl, concentration almost equivalent to sea water salinity), and that this occurs because, although the synthesis of ASP is not disturbed by the salt in the medium, the maturation pathway of serine protease of A. sobria (ASP) does not proceed successfully in such a medium. In this study, the effect of salt in the medium on the production of metalloprotease by A. sobria (AMP) was examined. A. sobria produced AMP in the milieu when the bacteria were cultured in medium containing (NaCl) at a concentration of 0.5%. However, AMP was not produced when the bacteria were cultured in salty medium containing 1.5% or more NaCl. To examine how NaCl reduces the production of metalloprotease by A. sobria, the amount of amp mRNA in the cell was measured and it was found that this decreased in proportion to the concentration of NaCl in the medium. The mRNA of amp was not detected in cells cultured in medium containing 1.5% or more NaCl. This means that the transcription of amp is inhibited in salty condition. As described, NaCl in the medium disturbs the maturation pathway of ASP. The mode of action whereby NaCl suppresses AMP activity in A. sobria differs from the mechanism for suppressing ASP activity.