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This study aimed to screen the inhalation toxicity of chemicals found in consumer products such as air fresheners, fragrances, and anti-fogging agents submitted to K-REACH using machine learning models. We manually curated inhalation toxicity data based on OECD test guideline 403 (Acute inhalation), 412 (Sub-acute inhalation), and 413 (Sub-chronic inhalation) for 1709 chemicals from the OECD eChemPortal database. Machine learning models were trained using ten algorithms, along with four molecular fingerprints (MACCS, Morgan, Topo, RDKit) and molecular descriptors, achieving F1 scores ranging from 51 % to 91 % in test dataset. Leveraging the high-performing models, we conducted a virtual screening of chemicals, initially applying them to data-rich chemicals generally used in occupational settings to determine the prediction uncertainty. Results showed high sensitivity (75 %) but low specificity (23 %), suggesting that our models can contribute to conservative screening of chemicals. Subsequently, we applied the models to consumer product chemicals, identifying 79 as of high concern. Most of the prioritized chemicals lacked GHS classifications related to inhalation toxicity, even though they were predicted to be used in many consumer products. This study highlights a potential regulatory blind spot concerning the inhalation risk of consumer product chemicals while also indicating the potential of artificial intelligence (AI) models to aid in prioritizing chemicals at the screening level., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Micro/nanoplastics (MNPs) have emerged as a significant environmental concern due to their widespread distribution and potential adverse effects on human health and the environment. In this study, to integrate exposure and toxicity pathways of MNPs, a comprehensive review of the occurrence, toxicokinetics (absorption, distribution, and excretion [ADE]), and toxicity of MNPs were investigated using the aggregate exposure pathway (AEP) and adverse outcome pathway (AOP) frameworks. Eighty-five papers were selected: 34 papers were on detecting MNPs in environmental samples, 38 papers were on the ADE of MNPs in humans and fish, and 36 papers were related to MNPs toxicity using experimental models. This review not only summarizes individual studies but also presents a preliminary AEP-AOP framework. This framework offers a comprehensive overview of pathways, enabling a clearer visualization of intricate processes spanning from environmental media, absorption, distribution, and molecular effects to adverse outcomes. Overall, this review emphasizes the importance of integrating exposure and toxicity pathways of MNPs by utilizing AEP-AOP to comprehensively understand their impacts on human and ecological organisms. The findings contribute to highlighting the need for further research to fill the existing knowledge gaps in this field and the development of more effective strategies for the safe management of MNPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Nanoplastics (NPs) are potentially toxic and pose a health risk as they can induce an inflammatory response and oxidative stress at cellular and organismal levels. Humans can be exposed to NPs through various routes, including ingestion, inhalation, and skin contact. Notably, uptake into the body via inhalation could result in brain accumulation, which may occur directly across the blood-brain barrier or via other routes. NPs that accumulate in the brain may be endocytosed into neurons, inducing neurotoxicity. Recently, we demonstrated that exposure to polystyrene (PS)-NPs reduces the viability of neurons. We have also reported that inhibiting the retrograde transport of PS-NPs by histone deacetylase 6 (HDAC6) prevents their intracellular accumulation and promotes their export in mouse embryonic fibroblasts. However, whether HDAC6 inhibition can improve neuronal viability by increasing exocytosis of PS-NPs from neurons remains unknown. In this study, mice were intranasally administered fluorescent PS-NPs (PS-YG), which accumulated in the brain and showed potential neurotoxic effects. In cultured neurons, the HDAC6 inhibitor ACY-1215 reduced the fluorescence signal detected from PS-YG, suggesting that the removal of PS-YG from neurons was promoted. Therefore, these results suggest that blocking the retrograde transport of PS-NPs using an HDAC6 inhibitor can alleviate the neurotoxic effects of PS-NPs that enter the brain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Recent studies have highlighted the potential of the ToxCast™ database for mechanism-based prioritization of chemicals. To explore the applicability of ToxCast data in the context of regulatory inventory chemicals, we screened 510 priority existing chemicals (PECs) regulated under the Act on the Registration and Evaluation, etc. of Chemical Substances (K-REACH) using ToxCast bioassays. In our analysis, a hit-call data matrix containing 298984 chemical-gene interactions was computed for 949 bioassays with the intended target genes, which enabled the identification of the putative toxicity mechanisms. Based on the reactivity to the chemicals, we analyzed 412 bioassays whose intended target gene families were cytochrome P450, oxidoreductase, transporter, nuclear receptor, steroid hormone, and DNA-binding. We also identified 141 chemicals based on their reactivity in the bioassays. These chemicals are mainly in consumer products including colorants, preservatives, air fresheners, and detergents. Our analysis revealed that in vitro bioactivities were involved in the relevant mechanisms inducing in vivo toxicity; however, this was not sufficient to predict more hazardous chemicals. Overall, the current results point to a potential and limitation in using ToxCast data for chemical prioritization in regulatory context in the absence of suitable in vivo data., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Living organisms adapt to their environment, and this adaptive response to environmental changes is influenced by both genomic and epigenomic components. As adaptation underpins tolerance to stressors, it is crucial to consider biological adaptation in evaluating the adverse outcomes of environmental chemicals, such as biocides. Daphnid studies have revealed differences in sensitivity to environmental chemicals between conspecific populations or clones, as well as between species. This study aimed to identify whether sensitivity to chemicals is subject to intraspecific variation, and whether this sensitivity depends on the genetic and epigenetic backgrounds of the daphnid population. We used an integrative approach to assess the comparative toxicity of a mixture of 5-chloro-2-methyl-4-isothiazoline-3-one and 2-methyl-4-isothiazolin-3-one (CMIT/MIT), a commonly used isothiazolinone biocide, by measuring mortality, reproduction, physiological traits, global DNA methylation, and proteomic expression at the species and strain levels. The results showed that the variation in sensitivity to CMIT/MIT between conspecific strains (Daphnia pulex; DPR vs. DPA strains) could exceed that observed between congeneric species (D. magna vs. D. pulex DPR strain). Under the control conditions, DPR (the strain most sensitive to CMIT/MIT) was characterized by a larger body size, a higher heart rate, and a higher level of global DNA methylation compared to its counterpart (DPA), and proteome profiles differed between the two strains. Particularly, the study identified strain-specific epigenetic and proteomic responses to LC20 of CMIT/MIT, demonstrating putative critical proteins and biological pathways associated with the observed differences in phenotype and sensitivity to CMIT/MIT. Downregulation of certain proteins (e.g., SAM synthase, GSTs, hemoglobin, and cuticle proteins) and DNA hypomethylation can be proposed as key events (KEs) of adverse outcome pathway (AOP) for isothiazolinone toxicity. Our findings indicate that both genetic variations and epigenetic modifications can lead to intraspecific variation in sensitivity to chemicals, and this variation should be considered in the ecological risk assessment framework for chemical substances. We suggest conducting further analysis on methylated gene regions and observing transgenerational effects to verify the role of crosstalk between genetic and epigenetic factors in phenotypic and protein expressions. DATA AVAILABILITY: Proteomic data is available in supplementary materials., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Jinhee Choi reports financial support was provided by National Research Foundation of Korea. Jiwan Kim reports financial support was provided by National Research Foundation of Korea. Marie-Agnes Coutellec reports financial support was provided by Hubert Curien., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

An adverse outcome pathway (AOP) framework can facilitate the use of alternative assays in chemical regulations by providing scientific evidence. Previously, an AOP, peroxisome proliferative-activating receptor gamma (PPARγ) antagonism that leads to pulmonary fibrosis, was developed. Based on a literature search, PPARγ inactivation has been proposed as a molecular initiating event (MIE). In addition, a list of candidate chemicals that could be used in the experimental validation was proposed using toxicity database and deep learning models. In this study, the screening of environmental chemicals for MIE was conducted using in silico and in vitro tests to maximize the applicability of this AOP for screening inhalation toxicants. Initially, potential inhalation exposure chemicals that are active in three or more key events were selected, and in silico molecular docking was performed. Among the chemicals with low binding energy to PPARγ, nine chemicals were selected for validation of the AOP using in vitro PPARγ activity assay. As a result, rotenone, triorthocresyl phosphate, and castor oil were proposed as PPARγ antagonists and stressor chemicals of the AOP. Overall, the proposed tiered approach of the database-in silico-in vitro can help identify the regulatory applicability and assist in the development and experimental validation of AOP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This study investigated the abundance and diversity of antibiotic resistance genes (ARGs) and the composition of bacterial communities along a transect covering the western Pacific Ocean (36°N) to the Southern Ocean (74°S) using the Korean icebreaker R/V Araon (total cruise distance: 14,942 km). The relative abundances of ARGs and bacteria were assessed with quantitative PCR and next generation sequencing, respectively. The absolute abundance of ARGs was 3.0 × 10 6 ± 1.6 × 10 6 copies/mL in the western Pacific Ocean, with the highest value (7.8 × 10 6 copies/mL) recorded at a station in the Tasman Sea (37°S). The absolute abundance of ARGs in the Southern Ocean was 1.8-fold lower than that in the western Pacific Ocean, and slightly increased (0.7-fold) toward Terra Nova Bay in Antarctica, possibly resulting from natural terrestrial sources or human activity. β-Lactam and tetracycline resistance genes were dominant in all samples (88-99%), indicating that they are likely the key ARGs in the ocean. Correlation and network analysis showed that Bdellovibrionota, Bacteroidetes, Cyanobacteria, Margulisbacteria, and Proteobacteria were positively correlated with ARGs, suggesting that these bacteria are the most likely ARG carriers. This study highlights the latitudinal profile of ARG distribution in the open ocean system and provides insights that will help in monitoring emerging pollutants on a global scale., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Dimethyl sulfide (DMS) produced by marine algae represents the largest natural emission of sulfur to the atmosphere. The oxidation of DMS is a key process affecting new particle formation that contributes to the radiative forcing of the Earth. In this study, atmospheric DMS and its major oxidation products (methanesulfonic acid, MSA; non-sea-salt sulfate, nss-SO 4 2- ) and particle size distributions were measured at King Sejong station located in the Antarctic Peninsula during the austral spring-summer period in 2018-2020. The observatory was surrounded by open ocean and first-year and multi-year sea ice. Importantly, oceanic emissions and atmospheric oxidation of DMS showed distinct differences depending on source regions. A high mixing ratio of atmospheric DMS was observed when air masses were influenced by the open ocean and first-year sea ice due to the abundance of DMS producers such as pelagic phaeocystis and ice algae. However, the concentrations of MSA and nss-SO 4 2- were distinctively increased for air masses originating from first-year sea ice as compared to those originating from the open ocean and multi-year sea ice, suggesting additional influences from the source regions of atmospheric oxidants. Heterogeneous chemical processes that actively occur over first-year sea ice tend to accelerate the release of bromine monoxide (BrO), which is the most efficient DMS oxidant in Antarctica. Model-estimates for surface BrO confirmed that high BrO mixing ratios were closely associated with first-year sea ice, thus enhancing DMS oxidation. Consequently, the concentration of newly formed particles originated from first-year sea ice, which was a strong source area for both DMS and BrO was greater than from open ocean (high DMS but low BrO). These results indicate that first-year sea ice plays an important yet overlooked role in DMS-induced new particle formation in polar environments, where warming-induced sea ice changes are pronounced., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The copper biotic ligand model (BLM) can quantitatively describe the bioavailability depending on various environmental factors and has been used to derive the predicted no-effect concentrations (PNECs). The commonly employed acute BLM tool, HydroQual, which applies the biotic ligand constants of fathead minnow in the same model structure for all taxonomic groups, estimates lower acute copper toxicity values compared to the chronic copper PNECs of the European Union Risk Assessment Reports (EU-RAR), which are based on taxon-specific model structures and biotic ligand constants for vertebrates, invertebrates, and algae. In this study, the full-BLM approach was applied using an appropriate acute BLM for each taxonomic group to derive acute HC5s (fifth percentile value in the species sensitivity distribution [SSD]) and an acute-chronic ratio for copper. Two acute BLMs for vertebrates and invertebrates were used and validated against site waters using the new method to estimate the intrinsic sensitivity for each species across different environmental conditions. To derive acute copper full BLM-based HC5s in Korean freshwater, acute toxicity tests were performed with 10 indigenous species, which were used to build the acute BLM-based SSD at each site. The final estimated acute full-BLM HC5s were higher than the EU-RAR chronic PNECs within the BLM calibration range. Furthermore, a linear relationship was observed between the acute full-BLM HC5s and the EU-RAR chronic PNECs. This linear regression function was suggested as an acute to chronic transformation function that can be applied to calculate chronic PNEC values. In conclusion, if the chronic ecotoxicity database of indigenous aquatic organisms for copper is lacking, it may be more efficient to derive chronic PNECs using an acute-chronic ratio after deriving BLM-based acute copper SSDs for indigenous species within representative taxonomic groups. This study provides a scientific foundation for the derivation of water quality criteria for copper in freshwater., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

To gain insight into the human health implications of microplastics, in this study, we investigated the possible mechanisms affecting the toxicity of high-density polyethylene (HDPE) in the nematode Caenorhabditis elegans using RNAi screening and a bioinformatics-based unbiased approach. The candidate pathways identified from C. elegans study were also confirmed using vertebrate model, zebrafish, Danio rerio and human relevance was then inferred using Comparative Toxicogenomics Database (CTD) analysis. Prior to evaluating the toxicity, label-free Raman mapping was conducted to investigate whether or not the organisms could uptake HDPE. C. elegans transcription factor RNAi screening results showed that the nucleotide excision repair (NER) and transforming growth factor-beta (TGF-β) signaling pathways were significantly associated with HDPE exposure, which was also confirmed in zebrafish model. Gene-disease interaction analysis using the CTD revealed the possible human health implications of microplastics. Finally, based on this finding, related AOPs were identified from AOP Wiki (http://aopwiki.org), which are "Peroxisome proliferator-activated receptors γ inactivation leading to lung fibrosis" and "AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma". Further studies are needed for the validation of these AOPs with various microplastics., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Microbial community of an organism plays an important role on its fitness, including stress responses. In this study, we investigated the effect of the culturable subset of soil microbial community (SMB) on the stress response of the soil nematode Caenorhabditis elegans, upon exposure to one of the major soil contaminants, cadmium (Cd). Life history traits and the stress responses to Cd exposure were compared between SMB- and Escherichia coli strain OP50-fed worms. SMB-fed worms showed higher reproduction rates and longer lifespans. Also, the SMB-fed worms showed more tolerant response to Cd exposure. Gene expression profiling suggested that the chemical stress and immune response of worms were boosted upon SMB feeding. Finally, we investigated C. elegans gut microbial communities in the presence and absence of Cd in OP50- and SMB-fed C. elegans. In the OP50-fed worms, changes in microbial community by Cd exposure was severe, whereas in the SMB-fed worms, it was comparatively weak. Our results suggest that the SMB affects the response of C. elegans to Cd exposure and highlight the importance of the gut microbiome in host stress response., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Silica nanoparticles (SiNPs) are one of the popular nanomaterials used in industrial manufacturing, synthesis, engineering, and medicine. Recently, mechanisms underlying toxicity of silica nanoparticles have been reported; however, their uptake mechanisms have still not fully understood. In this study, toxicity of SiNPs was investigated in the nematode Caenohabditis elegans by using microarray and pathway analysis focusing the uptake mechanisms and their impact on toxicity. Physicochemical characterization of SiNPs was performed using dynamic light scattering (DLS) and zeta potential. No mortality was observed after 24 h exposure to SiNPs. However, reproductive ability was significantly reduced at the same concentrations. To ascertain a global mechanism of toxicity, microarray was conducted on C. elegans exposed to 10 mg/L SiNPs (20% reduction in reproductive ability). Microarray results indicated that genes involved in reproduction, such as msp (Major Sperm Protein) genes, were significantly downregulated in C. elegans exposed to SiNPs. Pathway analyses on differentially expressed genes (DEGs) revealed that endocytic pathway as a major pathway involved in the uptake of SiNPs. Involvement of endocytic pathway in the uptake of SiNPs was assessed using specific inhibitors (methyl-β-cyclodextrin, chlorpromazine, and LY294002 for caveolin-, clathrin-, and pinocytosis-mediated endocytosis, respectively). The inhibitor assay indicated that an internalization process facilitated by clathrin-mediated endocytosis is involved in the uptake of SiNPs. Functional analysis using endocytosis defective mutants, (i,e.  cav-1, cup-2, and chc-1) confirmed the role of endocytosis on the reproductive toxicity of SiNPs. Overall results suggest that clathrin-mediated endocytosis pathway is a potential mechanism of uptake of SiNPs in C. elegans that in turn, affects general toxic outcome, such as, decrease in reproductive ability., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ecotoxicity tests conducted under well-controlled lab conditions often do not reflect the real environmental conditions. To this end, we designed an ecotoxicity test using an aquatic midge, Chironomus riparius, raised in metal-contaminated field sediments (MCFS), which reflect the real environmental conditions, for five consecutive generations (F0-F4) followed by a toxic response to arsenic exposure (as a second challenge). The toxic responses (i.e. DNA damage, DNA methylation, stress response gene expression, and mortality) were compared to those organisms reared in lab sediments (LS). Under the MCFS condition, increased adult emergence was observed for the second and third generations (F1 and F2), while a decreased tendency was evident thereafter (F3 and F4) compared to that of F0. When comparing C. riparius raised in MCFS or LS exposed to arsenic, increased sensitivity (declined survival) was observed in the larvae from F2. However, that tendency was not present in F4 of the MCFS midges, indicating a possible physiological adaptation. Increased DNA damage was observed in the MCFS-exposed organisms (F0, F2, and F4) compared to the those exposed to LS, particularly at F0. Arsenic exposure induced hypermethylation at F0 and, in contrast, hypomethylation at the later generations (F2, F4) in the MCFS-exposed organisms. Global DNA methylation results were supported by the expression of genes involved in enzymatic methylation. Moreover, alterations in oxidative stress related to gene expression showed that significant oxidative stress and perturbation of glutathione reserves occurred under the MCFS and the subsequent arsenic exposure conditions. Overall, our results suggest that multigenerational rearing under MCFS conditions resulted in physiological adaptation of C. riparius to metal exposure, specifically at later generations, which in turn modulated its response to arsenic stress through possible genetic and epigenetic mechanisms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Graphene oxide (GO) has the potential for wide applications, which necessitates an intensive investigation of its potential hazard on human and environmental health. Even if previous studies show reproductive toxicity in the nematode Caenorhabditis elegans, the mechanisms of reproductive toxicity by GO are poorly understood. To understand the underlying mechanisms of GO-induced reproductive toxicity, we investigated the interaction between GO and C. elegans using Raman spectroscopy, sperm counts produced by spermatogenesis, progeny and analyzed the fatty acid metabolism using molecular techniques. GO-characteristic Raman spectral bands measured throughout C. elegans, brood size and Hoecst staining of dissected gonads clearly showed GO accumulation in the reproductive organs, reduced progeny and low sperm counts, which are possibly direct results of the reproductive toxicity from GO exposure. Interestingly, reduced fatty acid metabolites, such as stearic, oleic, palmitoleic, and palmitic acids, were found with GO exposure. We found that GO increased intestinal fat accumulation in wild type N2, fat-5(tm420), and fat-7(wa36) mutants, whereas it decreased fat storage in the fat-6(tm331) and nhr-49(nr2041) mutants. GO exposure affected C. elegans fat accumulation and consumption, which was possibly regulated by daf-16 and nhr-80 gene activity. Also, GO exposure suppressed the survival of long-lived fat-5(tm420) mutants, whereas it increased the survival of short-lived nhr-49(nr2041) mutants. Hence, our studies collectively indicated that GO accumulation in reproductive organs, suppression of spermatogenesis, and the alteration of fatty acid metabolism play critical roles in understanding mechanisms of toxicity in C. elegans., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The stress response mechanisms of Bisphenol A (BPA), an endocrine disrupting compound, remain to be elucidated. In this study, we explored the effects of BPA on the non-biting midge Chironomus riparius through basic ecotoxicity assays, DNA damage (comet assay), eco-epigenetics (global DNA and histone methylations) and non-targeted global metabolomics (NMR based) approaches. The reproduction failure, increase in DNA damage, global DNA hyper-methylation, and increased global histone modification (H3K36) status were evident due to BPA exposure at 10% lethal concentration (LC 10 : 1 mg/L, based on 48 h acute toxicity). Moreover, non-targeted global metabolomics followed by pathway analysis identified alterations of energy metabolism, amino acids, and methionine metabolisms etc. Most importantly, we found a potential cross-talk between altered epigenetics and metabolites, such as, increase in methionine and o-phosphocholine metabolites corresponds with the phenomena of global hyper-methylation in DNA and H3K36 mark. Overall, our results suggests that the crosstalk of global metabolomics and epigenetic modification was fundamental of the underlying mechanisms in BPA-induced stress response in C. riparius., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The present study aimed to investigate the mechanisms involved in amorphous silica nanoparticles (aSiNPs)-mediated hepatotoxicity through the evaluation of changes in global metabolomics in in vitro and in vivo systems. 1 H NMR-based non-targeted global metabolomics and biochemical approaches were conducted in an aSiNPs-treated human hepatoma cell line (HepG2) and in ICR mice liver. The non-targeted NMR-based metabolomic analysis, followed by pathway analysis, revealed the perturbation of glutathione metabolism and the depletion of the glutathione pool after aSiNPs treatment in both in vitro (HepG2 cells) and in vivo systems. The total glutathione level, glutathione-S-transferase enzyme activity, and antioxidant gene expression strongly corroborated the metabolomic analysis results. The in vitro results were further supported by the in vivo data, specifically for metabolites profiling (Pearson Correlation coefficient is 0.462 (p = 0.026)). Furthermore, the depletion of glutathione, the formation of NADPH oxidase-mediated reactive oxygen species, and oxidative stress were evident in aSiNPs-treated HepG2 cells. Overall, the suppression of glutathione metabolism and oxidative stress are among the principal causes of aSiNPs-mediated hepatotoxicity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Reactive oxygen species (ROS) are strong oxidants, and have attracted considerable attention in both biological and environmental fields. Although various methods for ROS detection, including optical and electrochemical techniques, have been developed, they still face challenging issues in terms of poor sensitivity, reproducibility, stability, and in vivo applicability. Here, we present a sensitive and selective optical sensor for ROS detection, based on single plasmonic nanoprobes and redox-active cytochrome c (Cyt c)-mediated plasmon resonance energy transfer. By measuring the spectral changes of plasmonic nanoprobes, derived from the unique molecular absorption of Cyt c in accordance with the redox state, calibration curves for H 2 O 2 , a representative ROS, in various media were obtained over a wide concentration range from 100 mM to 1 nM. Limit of detection and limit of quantification in accordance with the used medium were determined from 8.3 to 12.8 nM and from 27.6 to 42.7 nM, respectively. Selectivity coefficients for major interfering solutes were much lower than 0.1 indicating a good selectivity for ROS. From the dynamic spectral changes, we sensitively monitored ROS generated in Caenorhabditis elegans (C. elegans) exposed to graphene oxide. Based on the calibration curves, we also determined ROS levels in various aquatic environments, such as river streams and a small pond, as a way of environmental monitoring. We believe that our approach could provide an avenue for achieving dynamic and sensitive monitoring of ROS in toxicological, biological, and environmental fields in the future., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Effects observed within one generation disregard potential detrimental effects that may appear across generations. Previously we have developed a two generation Daphnia magna reproduction test using the OECD TG 211 protocol with a few amendments, including initiating the second generation with third brood neonates produced from first generation individuals. Here we showed the results of an inter-laboratory calibration exercise among 12 partners that aimed to test the robustness and consistency of a two generation Daphnia magna reproduction test. Pyperonyl butoxide (PBO) was used as a test compound. Following experiments, PBO residues were determined by TQD-LC/MS/MS. Chemical analysis denoted minor deviations of measured PBO concentrations in freshly prepared and old test solutions and between real and nominal concentrations in all labs. Other test conditions (water, food, D. magna clone, type of test vessel) varied across partners as allowed in the OECD test guidelines. Cumulative fecundity and intrinsic population growth rates (r) were used to estimate "No observed effect concentrations "NOEC using the solvent control as the control treatment. EC 10 and EC- 50 values were obtained regression analyses. Eleven of the twelve labs succeeded in meeting the OECD criteria of producing >60 offspring per female in control treatments during 21days in each of the two consecutive generations. Analysis of variance partitioning of cumulative fecundity indicated a relatively good performance of most labs with most of the variance accounted for by PBO (56.4%) and PBO by interlaboratory interactions (20.2%), with multigenerational effects within and across PBO concentrations explaining about 6% of the variance. EC 50 values for reproduction and population growth rates were on average 16.6 and 20.8% lower among second generation individuals, respectively. In summary these results suggest that the proposed assay is reproducible but cumulative toxicity in the second generation cannot reliably be detected with this assay., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The properties, fate, and toxicity of silver nanoparticles (AgNPs) are readily modified in the environment. Thus, in order to predict the environmental impact of AgNPs, the toxicity test should be conducted to assess the interactions of AgNPs with environmental matrices. Dissolved organic matter (DOM) is known to mitigate AgNPs toxicity in natural systems, and it is also known that silver binds strongly to sulfur. Little is known, however, about the effect of sulfidation and to what extent it could compete with DOM in the sediment. We therefore investigated the effect of sulfide on a sediment dwelling organism, Chironomus riparius using ecotoxicity endpoints. We then investigated how sulfide and a combination of sulfide and DOM affect the toxicity of AgNPs in C. riparius. We also monitored the concentrations of silver in the water and sediment compartments, as well as in C. riparius tissue, in the presence and absence of sulfide. Finally, in order to investigate how sulfide and DOM affect the release of ions from AgNPs, we also monitored released Ag(+) in each treatment. In the presence of sulfide, AgNPs were found to be less toxic to C. riparius in acute and chronic endpoints than AgNPs alone, whereas DOM treatment did not modulate the toxicity of AgNPs. Sulfide treatment reduced the release of Ag(+) from AgNPs. Water-spiked AgNPs with sulfide were found to be more slowly incorporated into both sediment and larvae as compared to the AgNP alone. Overall, the results suggest that the presence of sulfide in sediment mitigates the ecotoxicity of AgNPs in C. riparius., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The widespread applications of graphene family nanomaterials (GFNs) raised the considerable concern over human health and environment. The cyto-genotoxic potentiality of GFNs has attracted much more attention, albeit the potential effects on the cellular epigenome remain largely unknown. The effects of GFNs on cellular genome were evaluated with single and double stranded DNA damage and DNA repair gene expressions while the effects on epigenome was accomplished by addressing the global DNA methylation and expression of DNA methylation machineries at non-cytotoxic to moderately cytotoxic doses in in vitro system. We used five different representatives of GFNs-pristine (GNP-Prist), carboxylated (GNP-COOH) and aminated (GNP-NH2) graphene nanoplatelets as well as single layer (SLGO) and few layer (FLGO) graphene oxide. The order of single stranded DNA damage was observed as GNP-Prist ≥ GNP-COOH>GNP-NH2≥FLGO>SLGO at 10mg/L and marked dose dependency was found in SLGO. The GFNs possibly caused genotoxicity by affecting nucleotide excision repair and non-homologus end joining repair systems. Besides, dose dependent increase in global DNA methylation (hypermethylation) were observed in SLGO/FLGO exposure and conversely, GNPs treatment caused hypomethylation following the order as GNP-COOH>GNP-NH2 ≥ GNP-Prist. The decrements of DNA methyltransferase (DNMT3B gene) and methyl-CpG binding domain protein (MBD1) genes were probably the cause of global hypomethylation induced by GNPs. Conversely, the de novo methylation through the up-regulation of DNMT3B and MBD1 genes gave rise to the global DNA hypermethylation in SLGO/FLGO treated cells. In general, the GFNs induced genotoxicity and alterations of global DNA methylation exhibited compounds type specificity with differential physico-chemical properties. Taken together, our study suggests that the GFNs could cause more subtle changes in gene expression programming by modulating DNA methylation status and this information would be helpful for their prospective use in biomedical field., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The increased volumes of carbon nanotubes (CNTs) being utilized in industrial and biomedical processes carries with it an increased risk of unintentional release into the environment, requiring a thorough hazard and risk assessment. In this study, the toxicity of pristine and hydroxylated (OH-) multiwall CNTs (MWCNTs) was investigated in the nematode Caenorhabditis elegans using an integrated systems toxicology approach. To gain an insight into the toxic mechanism of MWCNTs, microarray and proteomics were conducted for C. elegans followed by pathway analyses. The results of pathway analyses suggested endocytosis, phagocytosis, oxidative stress and endoplasmic reticulum (ER) stress, as potential mechanisms of uptake and toxicity, which were subsequently investigated using loss-of-function mutants of genes of those pathways. The expression of phagocytosis related genes (i.e. ced-10 and rab-7) were significantly increased upon exposure to OH-MWCNT, concomitantly with the rescued toxicity by loss-of-function mutants of those genes, such as ced-10(n3246) and rab-7(ok511). An increased sensitivity of the hsp-4(gk514) mutant by OH-MWCNT, along with a decreased expression of hsp-4 at both gene and protein level suggests that MWCNTs may affect ER stress response in C. elegans. Collectively, the results implied phagocytosis to be a potential mechanism of uptake of MWCNTs, and ER and oxidative stress as potential mechanisms of toxicity. The integrated systems toxicology approach applied in this study provided a comprehensive insight into the toxic mechanism of MWCNTs in C. elegans, which may eventually be used to develop an "Adverse Outcome Pathway (AOP)", a recently introduced concept as a conceptual framework to link molecular level responses to higher level effects., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

In our previous in vitro study of the toxicity on silver nanoparticles (AgNPs), we observed a dramatically higher sensitivity of Jurkat T cells to AgNPs than to Ag ions, and DNA damage and apoptosis were found to be involved in that toxicity. In this study, to understand underlying mechanism of different sensitivity of Jurket T cells to AgNPs and Ag ions, mRNA microarray and micro RNA microarray were concomitantly conducted on AgNPs and Ag ions exposed Jurkat T cells. Surprisingly only a small number of genes were differentially expressed by exposure to each of the silver (15 altered mRNA by AgNPs exposure, whereas 4 altered mRNA by Ag ions exposure, as determined 1.5-fold change as the cut-off value). miRNA microarray revealed that the expression of 63 miRNAs was altered by AgNPs exposure, whereas that of 32 miRNAs was altered by Ag ions exposure. An integrated analysis of mRNA and miRNA expression revealed that the expression of hsa-miR-219-5p, was negatively correlated with the expression of metallothionein 1F (MT1F) and tribbles homolog 3 (TRIB3), in cells exposed to AgNPs; whereas, the expression of hsa-miR-654-3p was negatively correlated with the expression of mRNA, endonuclease G-like 1 (EDGL1) in cells exposed to Ag ions. Network analysis were further conducted on mRNA-miRNA pairs, which revealed that miR-219-5p-MT1F and -TRIB3 pairs by AgNPs are being involved in various cellular processes, such as, oxidative stress, cell cycle and apoptosis, whereas, miR-654-3p and ENDOGL1 pair by Ag ions generated a much simpler network. The putative target genes of AgNPs-induced miR-504, miR-33 and miR-302 identified by Tarbase 6.0 are also found to be involved in DNA damage and apoptosis. These results collectively suggest that distinct epigenetic regulation may be an underlying mechanism of different sensitivity of Jurkat T cells to AgNPs and Ag ion. Further identification of putative target genes of DE miRNA by AgNPs and Ag ions may provide additional clues for the mechanism of differential toxicity. Overall results suggest that epigenetic mechanism is involved in toxicity of AgNPs and Ag ions in Jurkat T cells., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Although silver nanoparticles (AgNPs) have been reported to exert strong acute toxic effects on various cultured cells by inducing oxidative stress, the molecular mechanisms by which AgNPs-damaged cells are unknown. Because the endoplasmic reticulum (ER) may play an important role in the response to oxidative stress-induced damage and is quite sensitive to oxidative damage, we hypothesized that AgNPs may exert cytotoxic effects on cells by modulating ER stress. In our study, AgNPs resulted in cytotoxicity and apoptotic cell death when analyzing cell viability, DNA fragmentation and the apoptotic sub-G(1) population. Flow cytometry and confocal microscopy indicated that the cells were sensitive to AgNPs with respect to the induction of mitochondrial Ca(2+) overloading and enhancement of ER stress. AgNPs induced a number of signature ER stress markers, including phosphorylation of RNA-dependent protein kinase-like ER kinase (PERK) and its downstream eukaryotic initiation factor 2α, phosphorylation of inositol-requiring protein 1 (IRE1), splicing of ER stress-specific X-box transcription factor-1, cleavage of activating transcription factor 6 (ATF6) and up-regulation of glucose-regulated protein-78 and CCAAT/enhancer-binding protein-homologous protein (CHOP/GADD153). Down-regulation of PERK, IRE1 and ATF6 expression using siRNA significantly decreased AgNPs-induced the enhancement of ER stress. In addition, down-regulation of CHOP expression with siRNA CHOP attenuated AgNPs-induced apoptosis. Taken together, the present study supports an important role for the ER stress response in mediating AgNPs-induced apoptosis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Recently, we reported that silver nanoparticles (AgNPs) induced reactive oxygen species (ROS) generation and the resultant oxidative stress contributes to the cell damage associated with AgNPs. 8-Oxoguanine (8-oxoG) is sensitive marker of ROS-induced DNA damage. 8-Oxoguanine DNA glycosylase 1 (OGG1) is an important DNA repair enzyme that recognizes and excises 8-oxoG. The aim of the present study was to examine the effect of AgNPs-induced oxidative stress on OGG1 and to elucidate mechanisms underlying AgNPs toxicity. AgNPs decreased OGG1 mRNA and protein expression, resulting in decreased OGG1 activity. Decreased OGG1 activity in AgNPs-treated cells led to increased 8-oxoG levels. The transcription factor NF-E2-related factor 2 (Nrf2) is an important factor in the inducible regulation of OGG1. AgNPs treatment decreased nuclear Nrf2 expression, translocation into nucleus, and transcriptional activity of Nrf2. Extracellular regulated kinase (ERK) and protein kinase B (PKB, AKT), which are upstream of Nrf2, contribute to OGG1 expression. AgNPs attenuated both active forms of ERK and AKT protein expression, resulting in suppression of Nrf2 and decrease of OGG1 expression. These studies demonstrate that down-regulation of Nrf2-mediated OGG1 in exposure to AgNPs occurs through ERK and AKT inactivation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Silver nanoparticles (AgNPs), which have well-known antimicrobial properties, are extensively used in various medical and general applications. Despite the widespread use of AgNPs, relatively few studies have been undertaken to determine the cytotoxic effects of AgNPs exposure. This study investigates possible molecular mechanisms underlying the cytotoxic effects of AgNPs. Here, we show that AgNPs-induced cytotoxicity was higher compared than that observed when AgNO(3) was used as a silver ion source. AgNPs induced reactive oxygen species (ROS) generation and suppression of reduced glutathione (GSH) in human Chang liver cells. ROS generated by AgNPs resulted in damage to various cellular components, DNA breaks, lipid membrane peroxidation, and protein carbonylation. Upon AgNPs exposure, cell viability decreased due to apoptosis, as demonstrated by the formation of apoptotic bodies, sub-G(1) hypodiploid cells, and DNA fragmentation. AgNPs induced a mitochondria-dependent apoptotic pathway via modulation of Bax and Bcl-2 expressions, resulting in the disruption of mitochondrial membrane potential (Δψ(m)). Loss of Δψ(m) was followed by cytochrome c release from the mitochondria, resulting in the activation of caspases 9 and 3. The apoptotic effect of AgNPs was exerted via the activation of c-Jun NH(2)-terminal kinase (JNK) and was abrogated by the JNK-specific inhibitor, SP600125 and siRNA targeting JNK. In summary, the results suggest that AgNPs cause cytotoxicity by oxidative stress-induced apoptosis and damage to cellular components., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

To understand the molecular mechanism of previously observed cerium oxide (CeO(2)) nanoparticles-induced oxidative stress, an in vitro toxicity assay was conducted using human bronchial epithelial cell, Beas-2B, focusing on the involvement of the oxidative stress responding signal transduction pathway and transcription factors in the toxicity of CeO(2) nanoparticles. Extracellular signal-regulating kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) signaling pathways, along with nuclear factor-kappaB (NF-kappaB) and nuclear factor-E2-related factor-2 (Nrf-2), were investigated as the upstream events of oxidative stress from exposure to CeO(2) nanoparticles. The overall results suggest that CeO(2) nanoparticles may exert their toxicity through oxidative stress, as they cause significant increases in the cellular reactive oxygen species (ROS) concentrations, subsequently leading to the strong induction of heme oxygenase-1 (HO-1) via the p38-Nrf-2 signaling pathway. Further studies on the mechanism by which CeO(2) nanoparticles induce the p38-Nrf-2 signaling pathway are warranted for a better understanding of the CeO(2) nanoparticles-induced oxidative stress; studies with other signaling pathways, with concentration-response and time course experiments would also be justified.

To investigate the effects of chlorpyrifos (CP), an organophosphorus insecticide, on the soil nematode Caenorhabditis elegans, the toxicity of the insecticide on the molecular, biochemical, and physiological levels were investigated upon sublethal exposure, and an acute toxicity test was conducted using lethality as an endpoint. To assess the molecular-level effect, stress-related gene expression was investigated, and the neurotoxicity indicator, acetylcholinesterase (AChE) activity was assessed as the biochemical-level response. Growth, reproduction and development were also studied as physiological-level responses. The overall results indicate that CP exposure leads to the alteration of the expression of some stress genes, such as of heat shock protein, metallothionein, vitellogenin and C. elegans p53-like protein genes; the inhibition of AChE activity; and the retardation of development. These data suggest that the toxicity of CP on C. elegans occurred in multiple biological organizations; nevertheless this is not sufficient to conclude that there is a casual relationship between them. Thus, direct experimental demonstrations of the wider relationships between the molecular/biochemical effects of CP exposure and their consequences at higher levels of biological organization are needed to fully understand the effects of this compound on C. elegans.

As the applications of industrial nanoparticles are being developed, the concerns on the environmental health are increasing. Cytotoxicities of titanium dioxide nanoparticles of different concentrations (5, 10, 20 and 40 microg/ml) were evaluated in this study using a cultured human bronchial epithelial cell line, BEAS-2B. Exposure of the cultured cells to nanoparticles led to cell death, reactive oxygen species (ROS) increase, reduced glutathione (GSH) decrease, and the induction of oxidative stress-related genes such as heme oxygenase-1, thioredoxin reductase, glutathione-S-transferase, catalase, and a hypoxia inducible gene. The ROS increase by titanium dioxide nanoparticles triggered the activation of cytosolic caspase-3 and chromatin condensation, which means that titanium dioxide nanoparticles exert cytotoxicity by an apoptotic process. Furthermore, the expressions of inflammation-related genes such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), TNF-a, and C-X-C motif ligand 2 (CXCL2) were also elevated. The induction of IL-8 by titanium dioxide nanoparticles was inhibited by the pre-treatment with SB203580 and PD98059, which means that the IL-8 was induced through p38 mitogen-activated protein kinase (MAPK) pathway and/or extracellular signal (ERK) pathway. Uptake of the nanoparticles into the cultured cells was observed and titanium dioxide nanoparticles seemed to penetrate into the cytoplasm and locate in the peri-region of the nucleus as aggregated particles, which may induce direct interactions between the particles and cellular molecules, to cause adverse biological responses.

Cerium oxide nanoparticles of different sizes (15, 25, 30, 45 nm) were prepared by the supercritical synthesis method, and cytotoxicity was evaluated using cultured human lung epithelial cells (BEAS-2B). Exposure of the cultured cells to nanoparticles (5, 10, 20, 40 microg/ml) led to cell death, ROS increase, GSH decrease, and the inductions of oxidative stress-related genes such as heme oxygenase-1, catalase, glutathione S-transferase, and thioredoxin reductase. The increased ROS by cerium oxide nanoparticles triggered the activation of cytosolic caspase-3 and chromatin condensation, which means that cerium oxide nanoparticles exert cytotoxicity by an apoptotic process. Uptake of the nanoparticles to the cultured cells was also tested. It was observed that cerium oxide nanoparticles penetrated into the cytoplasm and located in the peri-region of the nucleus as aggregated particles, which may induce the direct interaction between nanoparticles and cellular molecules to cause adverse cellular responses.

This study was conducted to determine the toxic effects of bisphenol A (BPA) and ethynyl estradiol (EE), well-known endocrine disruptors, on Chironomus riparius under controlled laboratory conditions. Mortality, enzyme activities, and growth/development parameters were studied as acute, biochemical, and physiological toxicities, respectively. The results of the present study showed activation of catalase and glutathione-S-transferase after BPA and EE exposure, as well as increased emergence failure after EE exposure. This study on the effects of BPA and EE on C. riparius can be an important addition to the knowledge that has been obtained regarding the toxicology of BPA and EE in aquatic organism, on which limited data are available. The data obtained from this study, however, are not sufficient to establish any correlation or casual relationship between these two compounds and the response of C. riparius. Thus, further research is required to come up with direct experimental demonstrations of the wider relationship between the biochemical effects of BPA and EE on C. riparius and their consequences at higher levels of biological organization.

In this study, di(2-ethylhexyl)phthalate (DEHP) toxicities to Caenorhabditis elegans were investigated using multiple toxic endpoints, such as mortality, growth, reproduction and stress-related gene expression, focusing on the identification of chemical-induced gene expression as a sensitive biomarker for DEHP monitoring. The possible use of C. elegans as a sentinel organism in the monitoring of soil ecosystem health was also tested by conducting the experiment on the exposure of nematode to field soil. Twenty-four-hour median lethal concentration (LC50) data suggest that DEHP has a relatively high potential of acute toxicity to C. elegans. Decreases in body length and egg number per worm observed after 24h of DEHP exposure may induce long-term alteration in the growth and reproduction of the nematode population. Based on the result from the C. elegans genome array and indicated in the literatures, stress proteins, metallothionein, vitellogenin, xenobiotic metabolism enzymes, apoptosis-related proteins, and antioxidant enzyme genes were selected as stress-related genes and their expression in C. elegans by DEHP exposure was analyzed semi-quantitatively. Expression of heat shock protein (hsp)-16.1 and hsp-16.2 genes was decreased by DEHP exposure. Expression of cytochrome P450 (cyp) 35a2 and glutathione-S-transferease (gst)-4, phase I and phase II of xenobiotic metabolism enzymes, was increased by DEHP exposure in a concentration-dependent manner. An increase in stress-related gene expressions occurred concomitantly with the deterioration on the physiological level, which suggests an increase in expression of those genes may not be considered as a homeostatic response but as a toxicity that might have physiological consequences. The experiment with the soil from the landfill site suggests that the potential of the C. elegans biomarker identified in laboratory conditions should be calibrated and validated for its use in situ.

To identify potential biomarkers for the monitoring and risk assessment of benzo[a]pyrene (BaP), the oxidative stress-related DNA damage and p53 modification were investigated in human hepatoma HepG2 cells. Benzo[a]pyrene exposure induced a decrease in the cell viability, but increased the antioxidant enzyme activity as well as the DNA and lipid damage. The p53 protein activation appeared to have been a downstream response to the benzo[a]pyrene-induced DNA damage, suggesting p53 plays important roles in the defense against benzo[a]pyrene-induced genotoxicity. The response of phosphorylated p53 may be more sensitive towards benzo[a]pyrene exposure than normal p53. Following DNA damage, the activation of p53 acts as a transcriptional regulator of several target genes, including, p21 protein; a gene that encodes the Cdk inhibitor and is induced by exposure to benzo[a]pyrene. The p53 mRNA level was increased after the treatment of cells with benzo[a]pyrene, as well as following the induction of p53 protein, suggesting the benzo[a]pyrene-stimulated p53 accumulation may also be transcriptionally induced. The overall results suggest that benzo[a]pyrene leads to serious DNA damage, which leads to the transcription of the p53 gene; that the subsequent p53 protein accumulation up-regulates the cellular p21 protein. Oxidative DNA damage and p53 accumulation seem to be related to benzo[a]pyrene toxicity; however, their potential as biomarkers in environmental monitoring and risk assessment needs to be validated in the context of their specificity and sensitivity.

7,8-Dihydro-8-oxoguanine (8-oxo-Gua) and its nucleoside in cytosol are derived from the repair of oxidative DNA and the cleanup of oxidatively damaged DNA precursors, respectively. While the harmful effects of 8-oxo-Gua present in DNA have been studied extensively, few have reported its effects on cytosolic function. Our previous study showed that the addition of 8-oxo-dG to culture media caused an accumulation of 8-oxo-Gua in nuclear DNA in several leukemic cells including KG-1, which lack 8-oxoguanine glycosylase 1 (OGG1) activity due to mutational loss. However, the mechanism underlying 8-oxo-Gua level increases in DNA has not been addressed. In this study, we elucidated the metabolic fate of 8-oxo-Gua-containing nucleotide and the effect of exogenous 8-oxo-dG on DNA synthesis in KG-1 cells. We found that 8-oxo-dGMP was rapidly dephosphorylated to 8-oxo-dG rather than phosphorylated to 8-oxo-dGDP, thus indicating that 8-oxo-Gua-containing molecule is not used as a substrate for DNA synthesis in KG-1 cells. In fact, radiolabeled 8-oxo-dG was incubated but radioactivity was not detected in nuclear DNA of KG-1 cells, showing that 8-oxo-dG is not directly incorporated into DNA. Interestingly, the activity of DNA polymerase beta, which synthesize DNA with low fidelity increased in KG-1 cells treated with 8-oxo-dG, whereas the expression of DNA polymerase alpha decreased. In addition, the accumulation of 8-oxo-Gua in KG-1 DNA was completely inhibited by a specific inhibitor of DNA polymerase beta. Thus, our findings address that the insertion of 8-oxo-dG into KG-1 DNA is not due to the direct incorporation of exogenous 8-oxo-dG, but rather to the inaccurate incorporation of endogenous 8-oxo-dGTP by DNA polymerase beta. It further suggests that 8-oxo-dG in the cytosol may function as an active molecule itself and perturb the well-defined DNA synthesis.

Oxygen radicals attack guanine bases in DNA but they also attack cytoplasmic GTP forming 8-oxoGTP. The presence of 8-oxoGTP in cytoplasm is evidenced by the fact that cells contain MutT/MTH1 which hydrolyze 8-oxoGTP into 8-oxoGMP. In this study, the interaction between 8-oxoGTP and Ras, a small GTP-binding protein, was tested in vitro, and the action of 8-oxoGTP was compared to that of GTP. When purified Ras was treated with 8-oxoGTPgammaS, Ras was activated, as indicated by the enhanced binding of Ras with Raf-1. GTPgammaS also activated Ras but 8-oxoGTPgammaS had a much more potent effect. In lysates of human embryo kidney 293 cells, 8-oxoGTPgammaS activated not only Ras but also the downstream effectors of the Ras-ERK pathway, i.e., Raf-1 and ERK1/2. In contrast to Ras, other small GTP-binding proteins, Rac1 and Cdc42, were inactivated by 8-oxoGTPgammaS, whereas both of these proteins were activated by GTPgammaS, indicating that the biological natures of 8-oxoGTP and GTP differ. These results suggest the possibility that 8-oxoGTP is not a simple by-product but a functional molecule transmitting an oxidative signal to small GTP-binding proteins like Ras.

In this study, an attempt was made to develop a method to estimate oxidative damage of individual genes for assessing chemopreventive potential of dietary or medicinal plants. Oxidative damage was investigated on the two genes in gastric mucosal tissue infected with Helicobacter pylori, which were genes of glyceraldehydes-3-phosphate dehydrogenase (GAPDH), a house-keeping gene, and gene of insulin-like growth factor II receptor (IGFIIR), a gene known to be mutated frequently in gastric carcinoma. The oxidative damage in genomic DNA in the above tissue was confirmed by immunohistochemical study using monoclonal antibody to 8-hydroxyguanine (oh(8)G), which showed much higher degree of staining in their nuclei. Using the method we developed, it was demonstrated that the number of oh(8)G (indicated by 8-hydroxyguanine glycosylase (OGG1) sensitive sites) in GAPDH was almost not changed in H. pylori-infected tissue but in IGFIIR, it increased significantly. These results indicate that this method is valid for the estimate of oxidative damage of individual genes and also showed that the susceptibility of genomic DNA to attack of reactive oxygen species is not homogeneous but different depending upon the region of DNA. We expect to use this method in studies of carcinogenic mechanism and chemoprevention since it can provide more specific information pertaining to individual genes we are interested in., (Copyright 2003 Elsevier Science B.V.)