Genes and Environment最新文献

Background: When assessing the genotoxicity of substances containing probiotic candidates, such as lactic acid-producing bacteria, using the in vitro micronucleus test (MNT), bacterial growth in the test medium may reduce the pH of the medium. The low medium pH is known to induce cytotoxicity and false-positive results. In the TK6 cell system, it is difficult to completely remove the bacteria from the medium by washing post-treatment, leading to bacterial growth during the recovery period in the short-term treatment. In the present study, the low pH range yielding false positives in the TK6 cell MNT was investigated using media supplemented with acetic, lactic, or formic acids, which are non-genotoxic bacterial metabolites. Additionally, to suppress the bacterial growth during the recovery period using antibiotics, i.e., penicillin/streptomycin (P/S), gentamicin sulfate (GM), and amphotericin B (AP), the maximum applicable concentrations of them that did not affect TK6 cell growth or micronucleus induction were determined. Then, we conducted an MNT using a substance containing live lactic acid-producing bacteria to verify the effectiveness of the antibiotics.

Results: Acetic, lactic, and formic acids induced micronuclei in TK6 cells (false positive) at an initial pH of ≤ 6.2 and ≤ 6.0 in 3 h treatment with and without S9 mix, respectively, and of ≤ 6.7 in the continuous treatment. Media supplemented with P/S, GM, and AP did not affect TK6 cell growth or micronucleated cell frequencies in the negative and positive controls ≤ 400 unit/mL-400 µg/mL, ≤ 250, and ≤ 20 µg/mL, respectively. In an MNT with fermented milk containing live lactic acid-producing bacteria, supplementation with P/S or GM to media for the recovery cultures suppressed the bacterial growth, decreasing pH, and cytotoxicity.

Conclusion: This study revealed the low pH ranges yielding false positives in the TK6 cell MNT under short-term and continuous treatment conditions. These values will serve as references for interpreting the biological relevance of results. Under short-term treatment, optimal antibiotic supplementation in recovery cultures suppressed bacterial growth in the test substance and prevented the decrease in pH that could yield false positives. This approach might be useful for evaluating the genotoxicity of test substances containing probiotic candidates using the MNT.

Background: The rose ketone β-damascone (β-Dam) elicits positive results in the in vitro micronucleus (MN) assay using human lymphocytes, but shows negative outcomes in the Ames test and combined in vivo MN and comet assays. This has led to the interpretation that the in vitro MN result is a misleading positive result. Oxidative stress has been suggested as an indirect mode of action (MoA) for in vitro MN formation, with the α, β-unsaturated carbonyl moiety of the β-Dam chemical structure expected to cause misleading positive results through this MoA. In this study, we investigated the role of oxidative stress in β-Dam-induced in vitro MN formation by co-treatment with the antioxidant N-acetyl-L-cysteine (NAC), thereby highlighting a possible link between mitochondrial dysfunction and aneugenicity.

Results: β-Dam induced MN formation in both CHL/IU and BEAS-2B cells, with the response completely inhibited by co-treatment with NAC. Moreover, β-Dam induced oxidative stress-related reporter activity in the ToxTracker assay and increased reactive oxygen species levels, while decreasing glutathione levels, in BEAS-2B cells in the high-content analysis. All of these effects were suppressed by NAC co-treatment. These findings indicate that β-Dam elicits oxidative stress, which causes DNA damage and ultimately leads to MN induction. However, no significant DNA damage-related reporter activities were observed in the ToxTracker assay, nor was there an increased number of γH2AX foci in the high-content analysis. These data suggest that MN formation is not a DNA-reactive MoA. Considering recent reports of aneuploidy resulting from chromosome segregation defects caused by mitochondrial dysfunction, we investigated if β-Dam could cause such dysfunction. We observed that the mitochondrial membrane potential was dose-dependently impaired in BEAS-2B cells exposed to β-Dam.

Conclusions: These findings suggest that the oxidative stress induced by β-Dam exposure may be explained through an aneugenic MoA via mitochondrial dysfunction, thereby contributing to MN formation in mammalian cells.

Background: Primary aromatic amines (PAAs) present significant challenges in the prediction of mutagenicity using current standard quantitative structure activity relationship (QSAR) systems, which are knowledge-based and statistics-based, because of their low positive prediction values (PPVs). Previous studies have suggested that PAAs are metabolized into genotoxic nitrenium ions. Moreover, ddE, a relative-energy based index derived from quantum chemistry calculations that measures the stability nitrenium ions, has been correlated with mutagenicity. This study aims to further examine the ability of the ddE-based approach in improving QSAR mutagenicity predictions for PAAs and to develop a refined method to decrease false positive predictions.

Results: Information on 1,177 PAAs was collected, of which 420 were from public databases and 757 were from in-house databases across 16 laboratories. The total dataset included 465 Ames test-positive and 712 test-negative chemicals. For internal PAAs, detailed Ames test data were scrutinized and final decisions were made using common evaluation criteria. In this study, ddE calculations were performed using a convenient and consistent protocol. An optimal ddE cutoff value of -5 kcal/mol, combined with a molecular weight ≤ 500 and ortho substitution groups yielded well-balanced prediction scores: sensitivity of 72.0%, specificity of 75.9%, PPV of 65.6%, negative predictive value of 80.9% and a balanced accuracy of 74.0%. The PPV of the ddE-based approach was greatly reduced by the presence of two ortho substituent groups of ethyl or larger, as because almost all of them were negative in the Ames test regardless of their ddE values, probably due to steric hindrance affecting interactions between the PAA and metabolic enzymes. The great majority of the PAAs whose molecular weights were greater than 500 were also negative in Ames test, despite ddE predictions indicating positive mutagenicity.

Conclusions: This study proposes a refined approach to enhance the accuracy of QSAR mutagenicity predictions for PAAs by minimizing false positives. This integrative approach incorporating molecular weight, ortho substitution patterns, and ddE values, substantially can provide a more reliable basis for evaluating the genotoxic potential of PAAs.

Background: Although the in silico predictive ability of the Ames test results has recently made remarkable progress, there are still some chemical classes for which the predictive ability is not yet sufficient due to a lack of Ames test data. These classes include simple heterocyclic compounds. This study aimed to investigate the mutagenicity and structure-mutagenicity relationships for some heterocycles in the Ames test. In the present study, we selected 12 quinoline analogues containing one or two nitrogen atoms in the naphthalene ring and 12 indole analogues containing one to three nitrogen atoms in the indole ring, without any side moiety.

Results: The Ames test was performed with five standard bacterial strains (TA100, TA1535, TA98, TA1537, and WP2uvrA) using the pre-incubation method with and without rat liver S9. Five quinoline and two indole analogues were mutagenic. Among the five quinoline analogues, four were mutagenic in the presence of S9 mix with TA100, whereas cinnoline was mutagenic in the absence of S9 mix with TA1537. Among the two indole analogues, indazole was mutagenic in the presence and absence of S9 mix with WP2uvrA and 4-azaindole was mutagenic in the absence of S9 mix with TA1537. The mechanisms underlying the induction of mutagenesis appear to differ between quinoline and indole analogues. In addition, we performed in silico analysis of the mutagenicity of all these analogues using DEREK Nexus 6.1.1 (Lhasa Limited) and GT_EXPERT from CASE Ultra 1.8.0.5 (MultiCASE Inc.) as knowledge-based models and GT1_BMUT from CASE Ultra 1.8.0.5 (MultiCASE Inc.) as a statistical-based model. The knowledge-based model showed low sensitivity for both the quinoline and indole analogues (DEREK Nexus and GT_EXPERT: 20% for quinolines and 0% for indoles). Conversely, the statistical model showed high sensitivity (100% for both quinolines and indoles) and low specificity (43% for quinolines and 10% for indoles).

Conclusion: Based on the Ames test results, we proposed structural alerts noting that quinoline analogues were mutagenic when they had nitrogens in any of the positions 2, 5, 7, or 8 in addition to 1, and indole analogues were mutagenic when they had nitrogens at positions 2 or 4 in addition to 1.

Background: We previously investigated methods for separating mutagenic contaminants from aqueous solutions using cellulose-bearing covalently bound trisulfo-Cu-phthalocyanine (blue cotton and blue rayon). Mutagenic contaminants with three or more fused aromatic rings in their structures were adsorbed onto blue cotton and rayon. Since Cu-phthalocyanine is considered an unsuitable absorption ligand for byproducts of water chlorination, such as 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (Mutagen X or MX), we investigated the development of a new material for the elimination of MX from aqueous solvents.

Results: We selected green cellulose powder bearing ferrous phthalocyanine (FePh), hereafter referred to as green cellulose or GP, as the candidate material. GP is composed of cationized cellulose (white cellulose, WP) and FePh tetracarboxylic acid. The mutagenicity of MX dissolved in buffer or dimethyl sulfoxide (DMSO) solution significantly decreased after treatment with GP. The effects of GP on the elimination of MX from the solvent were very close to being expired after 70 cycles of repeated adsorption of the same GP, and the capacity of GP for MX removal was estimated to be exhausted after 120 cycles of repeated adsorption based on the extrapolation of the obtained result; thus, the interacting ligands on GP may be saturated after complete MX adsorption. The mutagenicity of MX dissolved in aqueous buffer significantly decreased after treatment at pH7.4 but not at pH 4.0. Since MX is dissociated to be the anionic form at pH 6 or higher, the negative charge of MX in the buffer at pH 7.4 may interact with the positive charge of ferrous ions in GP to create a linkage between MX and GP. After GP adsorbed MX, mutagenicity was extracted with water or acetonitrile and recovered in the eluent. Thus, the reversible interaction between MX and FePh may have caused adsorption of MX onto GP.

Conclusion: GP could be used as a new eliminator and recovery agent for MX in chlorinated drinking water. Developing new materials for the removal and recovery of agents for the detection of mutagenic contaminant-related chlorination in water is beneficial for environmental health.

Background: Both mutation induction and clonal expansion of mutated cells cause cancer. The probability of cancer development depends on mutations, clonal growth rates, and carcinogenic mechanisms. A recent study showed cases of occupational cholangiocarcinomas that originate multifocally, with higher mutation burden levels than those in common cholangiocarcinomas. This study aimed to identify the effect of clonal expansion on and estimate the risk of occupational and common intrahepatic cholangiocarcinomas (ICCs) using a multistage model modified to include the effect of cell expansion at any carcinogenic stage.

Methods: The age-specific incidence of common ICC estimated from the Vital Statistics in Japan and the prognosis of ICC, and mutation frequencies of occupational and common ICC available from the previous report, were applied to a multistage model modified with cell proliferation effects. From the fittest model, the risk after exposure was estimated.

Results: The required number of stages for carcinogenesis was estimated to be three based on the incidences and mutation frequencies of occupational and common ICCs. Based on this estimation, the predicted incidence curve under the model was similar to that estimated from the ICC mortality rate, except for older adults. The model indicated a minor effect of clonal expansion on the observed occupational ICC risk. It predicted a rapid decrease in ICC risk after the cessation of occupational exposure, although the time of clinical detection of cancer after the exposure was affected by latency. The model predicted an increase in cancer risk in older adults caused by cell expansion and common background mutations. However, the risk in older adults was overestimated in the case of common ICC; this divergence could influence occupational ICC cases.

Conclusions: Three-stage ICC carcinogenesis has been proposed. The high mutation burden levels caused by occupational exposure led to an immediate incidence of cancer. After a long period of relatively low cancer risk, an increased risk in older adults was also predicted.

Background: Error-corrected next-generation sequencing (ecNGS) technologies have enabled the direct evaluation of genome-wide mutations after exposure to mutagens. Previously, we reported an ecNGS methodology, Hawk-Seq™, and demonstrated its utility in evaluating mutagenicity. The evaluation of technical transferability is essential to further evaluate the reliability of ecNGS-based assays. However, cutting-edge sequencing platforms are continually evolving, which can affect the sensitivity of ecNGS. Therefore, the effect of differences in sequencing instruments on mutation data quality should be evaluated.

Results: We assessed the performance of four sequencing platforms (HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400) with the Hawk-Seq™ protocol for mutagenicity evaluation using DNA samples from mouse bone marrow exposed to benzo[a]pyrene (BP). The overall mutation (OM) frequencies per 10⁶ bp in vehicle-treated samples were 0.22, 0.36, 0.46, and 0.26 for HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400, respectively. The OM frequency of NextSeq2000 was significantly higher than that of HiSeq2500, suggesting the difference to be based on the platform. The relatively higher value in NextSeq2000 was a consequence of the G:C to C:G mutations in NextSeq2000 data (0.67 per 10⁶ G:C bp), which was higher than the mean of the four platforms by a ca. of 0.25 per 10⁶ G:C bp. A clear dose-dependent increase in G:C to T:A mutation frequencies was observed in all four sequencing platforms after BP exposure. The cosine similarity values of the 96-dimensional trinucleotide mutation patterns between HiSeq and the three other platforms were 0.93, 0.95, and 0.92 for NovaSeq, NextSeq, and DNBSeq, respectively. These results suggest that all platforms can provide equivalent data that reflect the characteristics of the mutagens.

Conclusions: All platforms sensitively detected mutagen-induced mutations using the Hawk-Seq™ analysis. The substitution types and frequencies of the background errors differed depending on the platform. The effects of sequencing platforms on mutagenicity evaluation should be assessed before experimentation.

Background: The Escherichia coli MutT (NudA) protein catalyzes the hydrolysis of an oxidized form of dGTP, 8-oxo-7,8-dihydro-dGTP (8-hydroxy-dGTP), and the spontaneous mutation frequency is elevated in E. coli cells deficient in the mutT gene.

Results: A split MutT, comprising the N-terminal (residues 1-95) and C-terminal (residues 96-129) peptides, was designed based on the known tertiary structure and linker insertion mutagenesis experiments. The mutator phenotype was complemented when the two peptides were separately expressed in mutT E. coli cells.

Conclusions: These results indicated that this split MutT functions as a nucleotide pool sanitization enzyme in vivo.

Background: Poly(ADP-ribose) polymerase-1 (PARP-1) is a pan nuclear protein that utilizes NAD⁺ as a substrate for poly(ADP-ribosyl)ation reaction (PARylation), resulting in both auto-modification and the modification of its accepter proteins. Earlier reports suggested that several nucleolar proteins interact and colocalize with PARP-1, leading to their PARylation. However, whether PARP-1 has any role in nucleolar biogenesis and the functional relevance of such a role is still obscure.

Results: Using PARP-1 depleted cells, we investigated the function of PARP-1 in maintaining the nucleolar morphology and protein levels under normal physiological conditions. Our results revealed that several nucleolar proteins like nucleolin, fibrillarin, and nucleophosmin get up-regulated when PARP-1 is depleted. Additionally, in line with the higher accumulation of nucleolin, stably depleted PARP-1 cells show lower activation of caspase-3, lesser annexin-V staining, and reduced accumulation of AIF in the nucleus upon induction of oxidative stress. Concurrently, PARP-1 silenced cells showed higher mitochondrial oxidative phosphorylation and more fragmented and intermediate mitochondria than the parental counterpart, suggesting higher metabolic activity for better survival.

Conclusion: Based on our findings, we demonstrate that PARP-1 may have a role in regulating nucleolar protein levels and mitochondrial activity, thus maintaining the homeostasis between cell protective and cell death pathways, and such cell-protective mechanism could be implicated as the priming state of a pre-cancerous condition or tumour dormancy.

Background: Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer commonly used in a wide variety of products, including medical devices. It is rapidly metabolized in the liver into various metabolites upon absorption through oral ingestion, dermal absorption, and inhalation. DEHP is classified as a non-genotoxic hepatocarcinogen in rodents, as its chronic exposure has been associated with the development of liver cancer in these animals, but most genotoxicity studies have been negative. Epidemiologic studies in humans suggest that long-term high intakes of DEHP may be a risk factor for liver dysfunction. The repeated-dose liver micronucleus (RDLMN) assay is a well-established method for assessing chromosomal changes caused by hepatic genotoxins and/or carcinogens. It is particularly valuable for detecting substances that undergo metabolic activation, especially when the metabolite has a short half-life or does not reach the bone marrow effectively. Therefore, we investigated whether the RDLMN assay could detect DEHP-induced micronucleus formation in the liver following a 14 or 28-day treatment.

Results: We report that the RDLMN assay demonstrated an increased frequency of hepatic micronuclei in rats exposed to DEHP for 14 or 28 days. The increases in micronuclei correlated with hepatomegaly, an established response to phthalates in the liver. Conversely, no such increases were observed in the micronucleus assay using bone marrow from these rats.

Conclusion: The detection of DEHP-induced micronuclei by the RDLMN assay suggests that this assay could detect the potential genotoxicity and hepatocarcinogenicity of DEHP. It also demonstrated the utility of the RDLMN assay in identifying metabolically activated hepatic carcinogens.