Impact of the Oxidant Type on the Efficiency of the Oxidation and Removal of Iron Compounds from Groundwater Containing Humic Substances.

Due to the coexistence of organic matter and iron in groundwater, a certain part of the iron is present as iron-organic complexes in the form of colloids and/or dissolved complexes. The study was conducted to evaluate the impact of the type of oxidizing agent: O2, Cl2, H2O2, or KMnO4, on the efficiency of the oxidation and removal of iron compounds from three groundwaters with significantly different contents and types of organic substances among which humic and fulvic acids occurred. This study shows that after the aeration and the oxidation with Cl2 and H2O2, the increasing content of dissolved hydrophilic organic substances containing aromatic rings in the raw water reduced the effectiveness of Fe(II) oxidation and the effectiveness of iron removal during the sedimentation process. This regularity was not found only when KMnO4 was used as the oxidant. After oxidation with H2O2, the highest number of organo-iron complexes and an increased concentration of dissolved organic carbon were found. High concentrations of organo-ferrous connections were also found in aerated water samples. The highest KMnO4 efficiency of removing iron and organic substances and reducing the color intensity and turbidity was due to the catalytic and adsorptive properties of the precipitated MnO2, which also improved the sedimentation properties of the resultant oxidation products.

Transcriptomic analysis of HepG2 cells exposed to fractionated wastewater effluents suggested humic substances as potential inducer of whole effluent toxicity.

We performed a transcriptome-based bioassay (TSB assay) using human hepatoma HepG2 cells to evaluate the potential toxicity of whole wastewater effluents from two membrane bioreactors (MBRs) and a conventional activated sludge process (AS). The biologically active agent(s) in the wastewater effluents were characterized based on expression of the marker genes (i.e., CYP1A1, AKR1B10, GCLM and GPX2) selected by DNA microarray analysis, after the wastewater effluent samples were concentrated by a reverse osmosis (RO) membrane and further fractionated by various manipulations. The qPCR assay of marker genes demonstrated that the induction of CYP1A1 and GPX2 was mitigated after passing through C18 and chelate columns. In addition, clear induction of CYP1A1 was observed in the smallest size fraction with 1 k Da or smaller organic molecules in all the tested effluents. These results together with the water quality data of the fractionated samples suggested that responsible constituents for potentially adverse and abnormal transcriptomic responses in HepG2 could have hydrophobic nature and act with metal-dissolved organic matter (DOM) complexes in 1 k Da or smaller size fraction. Although DOM is known to play two contradictory roles as a protector and an inducer of toxicants, our present study indicated the DOM in wastewater effluent, particularly humic substances with acidic nature, functioned as a toxicity inducer of residual chemicals in the effluents. This study provided a new insight into the nature of "toxic unknowns" in the wastewater effluents, which should be monitored whole through the reclamation process and prioritized for removal.

Detection of DNA damage formation by natural organic matter using EGFP-fused MDC1-expressing cells.

Studies have been conducted on the genotoxicity and carcinogenicity of disinfection by-products formed from natural organic matter (NOM) and mitigation effect for mutagens and clastogens by NOM. Whereas, reportedly, synthetic humic acid in high concentration has induced genotoxicity in human cells, and NOM samples have provoked mild oxidative and other physiological responses in aquatic organisms. Our group developed a novel detection method for DNA damage formation, namely enhanced green fluorescent protein (EGFP)-fused mediator of DNA damage checkpoint 1 (MDC1)-expressing human cells as simple and high-sensitive system. By using this method, a significant increase in the foci area was observed after 3 h, but not 24 h for 130 mgC L-1 Suwannee River fulvic acid (SRFA), 38 mgC L-1 humic acid (SRHA), and 19 mgC L-1 NOM (SRNOM). The SRNOM concentration is the original environmental one; therefore, it was suggested that the formation and repair of DNA damage associated with γ-H2AX, a biomarker for DNA double-strand breaks by mild oxidative stress, in Suwannee River (SR) were detected for the first time. The increase in the foci area was not observed for 18 mgC L-1 Lake Biwa fulvic acid (LBFA) and 50 mg L-1 catechin after both 3 h and 24 h. The difference between the SR and Lake Biwa (LB) samples may result from the differences in their electron-accepting capacity. The application of this methodology is expected to elucidate oxidative stress and toxicological effects shortly and in detail for many water samples.

Identification of oxidative stress and estrogenic activity induced by polarity fractions of effluent organic matter.

This study evaluated oxidative stress and estrogenic activity induced by hydrophobic (HP), transphilic (TP), and hydrophilic (HL) fractions of effluent organic matter (EfOM) from municipal and industrial wastewater treatment plants. Fluorescence regional integration (FRI) analysis indicated that EfOM showed higher FRI distribution in regions II (aromatic protein-like) and IV (soluble microbial products, SMPs) than Suwannee River natural organic matter (SRNOM), which was primarily in regions III (fulvic acid-like) and V (humic acid-like). The HP and HL fractions of industrial EfOMs significantly increased catalase (CAT) and glutathione S-transferase (GST) activity and lipid peroxidation compared with those of the control (p < 0.05). Estrogenic activity was the highest in industrial EfOM followed by municipal EfOM, and both were significantly higher than that of SRNOM and the control (p < 0.05). FRI distribution in region II was significantly related to CAT, GST, and estrogenic activity (r = 0.7142, 0.7786, and 0.8107, respectively) for the HL fraction of EfOM. For the HP fraction of EfOM, regions II and IV were significantly related to estrogenic activity (r = 0.7221 and 0.6707, respectively). These findings suggest that aromatic protein-like substances and SMPs in EfOM were responsible for the observed oxidative stress and estrogenic activity.

Influence of humic substances on the toxic effects of cadmium and SDBS to the green alga Scenedesmus obliquus.

The joint toxicity of chemicals mixture in the aquatic environment was still not well clear. To clarify the joint toxicity of the mixtures of metals and organic pollutants, as well as the influence of dissolved organic matter (DOM) in field water-body on their toxic effects, we conducted the toxicity tests with cadmium (Cd) and sodium dodecyl benzene sulfonate (SDBS) on Scenedesmus obliquus (S. obliquus) with or without the presence of fulvic acid (FA), a typical of DOM. Our results showed Cd was more toxic to S. obliquus than SDBS, and the effects of fulvic acid on SDBS were greater than Cd. The joint toxicity of Cd and SDBS expressed a synergistic effect on S. obliquus, which was observed to be increased with the presence of FA. Our results gave an example for the joint toxicity investigations of organics and metals, aiding to understanding the toxicity of pollutant mixtures in field water bodies containing DOM.

Nanoecotoxicity assessment of graphene oxide and its relationship with humic acid.

The risk assessment of nanomaterials is essential for regulatory purposes and for sustainable nanotechnological development. Although the application of graphene oxide has been widely exploited, its environmental risk is not well understood because several environmental conditions can affect its behavior and toxicity. In the present study, the graphene oxide effect from aquatic ecosystems was assessed considering the interaction with humic acid on 9 organisms: Raphidocelis subcapitata (green algae), Lemna minor (aquatic plant), Lactuca sativa (lettuce), Daphnia magna (planktonic microcrustacean), Artemia salina (brine shrimp), Chironomus sancticaroli (Chironomidae), Hydra attenuata (freshwater polyp), and Caenorhabditis elegans and Panagrolaimus sp. (nematodes). The no-observed-effect concentration (NOEC) was calculated for each organism. The different criteria used to calculate NOEC values were transformed and plotted as a log-logistic function. The hypothetical 5 to 50% hazardous concentration values were, respectively, 0.023 (0.005-0.056) and 0.10 (0.031-0.31) mg L-1 for graphene oxide with and without humic acid, respectively. The safest scenario associated with the predicted no-effect concentration values for graphene oxide in the aquatic compartment were estimated as 20 to 100 µg L-1 (in the absence of humic acid) and 5 to 23 µg L-1 (in the presence of humic acid). Finally, the present approach contributed to the risk assessment of graphene oxide-based nanomaterials and the establishment of nano-regulations. Environ Toxicol Chem 2018;37:1998-2012. © 2018 SETAC.

Interactions of natural organic matter on the surface of PVP-capped silver nanoparticle under different aqueous environment.

It is now widely accepted that coating on the nano-surface would critically dictate the uptake and cytotoxicity of engineering nanomaterials. However, the influence of natural organic matter (NOM) on the surface is quite limited to humic substances, while the diversity of NOM is neglected. In the present study, we tried to investigate the change of surface in the coexistence of bovine serum albumin (BSA) and humic acid (HA). The isothermal titration calorimetric measurements show that HA can combine with BSA in both freshwater or seawater, however, the patterns are different. In freshwater, HA lowered the adsorption of BSA on PVP-capped AgNPs through complexation with BSA, which prevented the contact of sulfur in BSA with PVP-AgNPs. Then in seawater, BSA retained its sulfur availability to bind with PVP-AgNPs. Furthermore, the toxicity of PVP-AgNPs incubated in the BSA/HA solution was evaluated by measuring the level of reactive oxygen species generated by Escherichia coli. The results indicated that, in seawater, the adsorbed BSA promoted the toxicity of PVP-AgNPs in the presence of Ca2+ and Mg2+, but the presence of HA limited this effect.

Effects of humic acids from landfill leachate on plants: An integrated approach using chemical, biochemical and cytogenetic analysis.

Biological process treatment of landfill leachate produces a significant amount of sludge, characterized by high levels of organic matter from which humic acids are known to activate several enzymes of energy metabolism, stimulating plant growth. This study aimed to characterize humic acids extracted from landfill sludge and assess the effects on plants exposed to different concentrations (0.5, 1, 2 and 4 mM C L-1) by chemical and biological analysis, to elucidate the influence of such organic material and minimize potential risks of using sludge in natura. Landfill humic acids showed high carbon and nitrogen levels, which may represent an important source of nutrients for plants. Biochemical analysis demonstrated an increase of enzyme activity, especially H+-ATPase in 2 mM C L-1 landfill humic acid. Additionally, cytogenetic alterations were observed in meristematic and F1 cells, through nuclear abnormalities and micronuclei. Multivariate statistical analysis provided integration of physical, chemical and biological data. Despite all the nutritional benefits of humic acids and their activation of plant antioxidant systems, the observed biological effects showed concerning levels of mutagenicity.

Time-dependent uptake and toxicity of nickel to Enchytraeus crypticus in the presence of humic acid and fulvic acid.

The present study aimed to investigate the influence of different fractions of dissolved organic carbon (DOC) on the uptake and toxicity of nickel (Ni) in the soil invertebrate Enchytraeus crypticus after different exposure times. The addition of DOC as humic acid or fulvic acid significantly reduced Ni uptake by E. crypticus in the soil-solution test system. Median lethal effect concentrations were calculated based on total dissolved Ni concentrations (LC50[Ni]), free Ni ion activity (LC50{Ni2+ }), and Ni body concentrations (LC50Body-Ni ). The LC50[Ni] values increased with increasing DOC levels and decreased with exposure time (4, 7, and 10 d). Humic acid exerted a greater protective effect on Ni toxicity than fulvic acid, but the protective effects decreased with prolonged exposure time. The LC50{Ni2+ } values also decreased with exposure time but were almost constant with variation in DOC levels, indicating that the protective effect of DOC is mainly through complexation with free Ni ions to reduce Ni bioavailability. The LC50Body-Ni value was independent of DOC concentration and exposure time, with an estimated overall value of 22.1 µg/g dry weight. The present study shows that body concentration could serve as an effective indicator for predicting Ni toxicity with variations in the exposure environment (e.g., DOC) and exposure time. Environ Toxicol Chem 2017;36:3019-3027. © 2017 SETAC.

Environmentally relevant concentration of arsenic trioxide and humic acid promoted tumor progression of human cervical cancer cells: In vivo and in vitro studies.

In a previous study, treatment at higher concentrations of arsenic trioxide or co-exposure to arsenic trioxide and humic acid was found to be inhibited cell growth of cervical cancer cells (SiHa cells) by reactive oxygen species generation. However, treatment at lower concentrations slightly increased cell viability. Here, we investigate the enhancement of progression effects of environmentally relevant concentration of humic acid and arsenic trioxide in SiHa cell lines in vitro and in vivo by measuring cell proliferation, migration, invasion, and the carcinogenesis-related protein (MMP-2, MMP-9, and VEGF-A) expressions. SiHa cells treated with low concentrations of humic acid and arsenic trioxide alone or in co-exposure significantly increased reactive oxygen species, glutathione levels, cell proliferation, scratch wound-healing activities, migration abilities, and MMP-2 expression as compared to the untreated control. In vivo the tumor volume of either single drug (humic acid or arsenic trioxide) or combined drug-treated group was significantly larger than that of the control for an additional 45 days after tumor cell injection on the back of NOD/SCID mice. Levels of MMP-2, MMP-9, and VEGF-A, also significantly increased compared to the control. Histopathologic effects of all tumor cells appeared round in cell shape with high mitosis, focal hyperkeratosis and epidermal hyperplasia in the skin, and some tumor growth in the muscle were observed. Our results may indicate that exposure to low concentrations of arsenic trioxide and humic acid is associated with the progression of cervical cancer. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1121-1132, 2016.

Humic Acid Increases Amyloid ß-Induced Cytotoxicity by Induction of ER Stress in Human SK-N-MC Neuronal Cells.

Humic acid (HA) is a possible etiological factor associated with for several vascular diseases. It is known that vascular risk factors can directly increase the susceptibility to Alzheimer's disease (AD), which is a neurodegenerative disorder due to accumulation of amyloid ß (Aß) peptide in the brain. However, the role that HA contributes to Aß-induced cytotoxicity has not been demonstrated. In the present study, we demonstrate that HA exhibits a synergistic effect enhancing Aß-induced cytotoxicity in cultured human SK-N-MC neuronal cells. Furthermore, this deterioration was mediated through the activation of endoplasmic reticulum (ER) stress by stimulating PERK and eIF2α phosphorylation. We also observed HA and Aß-induced cytotoxicity is associated with mitochondrial dysfunction caused by down-regulation of the Sirt1/PGC1α pathway, while in contrast, treating the cells with the ER stress inhibitor Salubrinal, or over-expression of Sirt1 significantly reduced loss of cell viability by HA and Aß. Our findings suggest a new mechanism by which HA can deteriorate Aß-induced cytotoxicity through modulation of ER stress, which may provide significant insights into the pathogenesis of AD co-occurring with vascular injury.

Mixture toxicity of nickel and zinc to Daphnia magna is noninteractive at low effect sizes but becomes synergistic at high effect sizes.

To incorporate metal mixture toxicity effects into risk-assessment procedures, more information is needed about combined and interactive effects of metal mixtures during chronic exposure. The authors investigated the toxicity of binary Ni-Zn mixtures in 2 independent full-factorial experiments using standard chronic (21-d) Daphnia magna reproduction toxicity tests. Global statistical analysis (i.e., when considering all investigated mixture treatments simultaneously) showed noninteractive effects according to the concentration addition model and significant synergistic effects according to the independent action model. However, treatment-specific statistical analysis revealed that both occurrence and type of interactive effect were dependent on the effect size at which Ni and Zn were combined in the mixture. Only noninteractive or weakly antagonistic effects occurred in mixture treatments in which each of the individual metals produced only weak adverse effects on its own (i.e., ≤20% reduction of reproductive performance). On the other side of the spectrum, synergistic mixture effects occurred in all mixture treatments where both metals already caused a > 20% (for independent action) and a > 40% (for concentration addition) effect on reproduction on their own. Because low effect sizes are the most relevant in most regulatory frameworks, the authors' data suggest that the concentration addition and independent action mixture toxicity models can both serve as conservative models for predicting effects of Ni-Zn mixtures. The present study highlights the importance of investigating metal mixture toxicity at low effect sizes and warns against extrapolating conclusions about metal mixture interactions from high to low effect sizes.

Induction of macrophage cell-cycle arrest and apoptosis by humic acid.

Humic acid (HA) in well water is associated with Blackfoot disease and various cancers. Previously, we reported that acute humic acid exposure (25-200 µg/mL for 24 hr) induces inflammation in RAW264.7 macrophages. In this study, we observed that prolonged (72 hr) HA exposure (25-200 µg/mL) induces cell-cycle arrest and apoptosis in cultured RAW264.7 cells. We also observed that exposing macrophages to HA arrests cells in the G2 /M phase of the cell cycle by reducing cyclin A/B1 , Cdc2, and Cdc25C levels. Treating macrophages with HA triggers a sequence of events characteristic of apoptotic cell death including loss of cell viability, morphological changes, internucleosomal DNA fragmentation, sub-G1 accumulation. Molecular markers of apoptosis associated with mitochondrial dysfunction were similarly observed, including cytochrome c release, caspase-3 or caspase-9 activation, and Bcl-2/Bax dysregulation. In addition to the mitochondrial pathway, HA-induced apoptosis may also be mediated through the death receptor and ER stress pathways, as evidence by induction of Fas, caspase-8, caspase-4, and caspase-12 activity. HA also upregulates p53 expression and causes DNA damage as assessed by the comet assay. These findings yield new insight into the mechanisms by which HA exposure may trigger atherosclerosis through modulation of the macrophage-mediated immune system.

Mechanism of the toxicity induced by natural humic acid on human vascular endothelial cells.

Humic acid (HA), a group of high-molecular weight organic compounds characterized by an ability to bind heavy metals, is normally found in natural water. Although the impairment of vascular endothelial cells in the presence of humic substances has been reported to be involved in some diseases, the mechanisms responsible for this involvement remain unclear. In this study, we examined the cytotoxicity of HA obtained from peatland in Central Kalimantan, Indonesia, to human vascular endothelial cells, as well as the mechanisms behind these effects. It was found that 50 mg/L HA showed cytotoxicity, which we considered to be mediated by apoptosis through the mitochondrial pathway because of an increase in the expression of caspases 6 and 9 in response to HA administration. In addition, this cytotoxicity was enhanced when cells in this experimental system were exposed to oxidative stress, while it was decreased by the addition of vitamin C. Thus, we conclude that the apoptosis induced by HA depends upon oxidative stress. Furthermore, an iron chelator, DFO, showed a tendency to decrease HA-induced cytotoxicity, suggesting that iron may potentially mediate HA-induced oxidative stress. In conclusion, long-term consumption of HA-rich water obtained from our study area may cause damage to endothelial cells and subsequent chronic health problems.

Antiandrogenic activity of humic substances.

For long, natural organic matter (NOM) composed mainly of humic substances (HS) were regarded as inert in the ecosystems with respect to their possible chemical interaction with exposed organisms. However recently, NOM have been shown to elicit various adverse effects generally attributed to synthetic xenobiotics, including estrogenic effects translating into shifts of the gender ratios in populations. However, the anti/androgenic pathway was not yet evaluated. Here, we applied an anti/androgenic sensitive cell line MDA stably transfected with the firefly luciferase gene under transcriptional control of the androgen responsive element. With five out of twelve tested NOM preparations, particularly with soil and coal isolates, we identified a relatively high, concentration-dependent antiandrogenic effect. This appears to be the first study to show this endocrine disrupting pathway for a ubiquitous biogeochemical matrix, a potential activity which should not be neglected in forthcoming studies on synthetic endocrine disruptors in the environment.

Humic acid enhances cigarette smoke-induced lung emphysema in mice and IL-8 release of human monocytes.

UNLABELLED: Tobacco smoke is the main factor in the etiology of lung emphysema. Generally prolonged, substantial exposure is required to develop the disease. Humic acid is a major component of cigarette smoke that accumulates in smokers' lungs over time and induces tissue damage. OBJECTIVES: To investigate whether humic acid pre-loading potentiates the development of cigarette smoke-induced lung emphysema in mice and increases IL-8 release by human monocytes. METHODS: C57BL/6J mice received humic acid or aqueous vehicle by tracheal installation on day 0 and day 7. From day 21 to day 84, the mice were exposed to cigarette smoke or clean air for 5 days/week. Twenty-four hours after the last exposure we determined leukocytes in lung lavage, heart hypertrophy and alveolar wall destruction. Human monocytes were incubated with cigarette smoke extract (CSE), humic acid or the combination overnight. RESULTS: Humic acid nor cigarette smoke caused alveolar wall destruction within two months. Interestingly, the combination did induce lung emphysema. Humic acid, cigarette smoke or the combination did not change leukocyte types and numbers in lung lavage fluid, but the combination caused peribronchiolar and perivascular lymphocyte infiltration. Humic acid treatment resulted in a high proportion of alveolar macrophages heavily loaded with intracellular granula. Humic acid also induces the release of IL-8 from human monocytes and enhances the CSE-induced IL-8 release. CONCLUSIONS: Humic acid deposition in the lungs potentiates the development of cigarette smoke-induced interstitial inflammation and lung emphysema. Moreover, humic acid promotes IL-8 release from human monocytes. Since humic acid accumulates steadily in the lungs of smokers, this may provide an explanation for the natural history on late onset of this disease. The model described here offers a novel way to study emphysema and may direct the search for new therapeutic approaches.

In vitro assessment of AhR-mediated activities of TCDD in mixture with humic substances.

Humic substances (HS) are ubiquitous natural products of decomposition of dead organic matter. HS is present in most freshwaters at concentrations ranging from 0.5 to 50mg L(-1). Organic carbon can represent 20% dry weight of sediments. Recently, the interaction of dissolved HS with the aryl hydrocarbon receptor (AhR) has been demonstrated. The AhR is a cytosolic receptor to which persistent organic pollutants (POPs) can bind and many of their toxic effects are mediated through interactions with this receptor. We describe in vitro effects (using H4IIE-luc cells) of binary mixtures of various HS with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), since in contaminated environments these compounds occur simultaneously. Six out of 12 HS samples activated AhR even at environmentally relevant concentrations (17 mg L(-1)), but did not reach the full AhR-activation even at excessive concentration. In simultaneous exposure of H4IIE-luc to HS (17 mg L(-1)) and TCDD (1.2 pM) without any preincubation prior to exposure, either significant additive or facilitative effects were observed. No negative interactions, due to possible sorption of TCDD to HS was observed. Nevertheless, if the HS-TCDD binary mixture was preincubated for 6 days prior to the exposure on H4IIE-luc cells, the additive and facilitative effects were less due to possible sorption of TCDD onto HS. Similar results were obtained from analogous experiments with greater concentrations of both TCDD and HS.

Humic acid induces G1 phase arrest and apoptosis in cultured vascular smooth muscle cells.

Humic acid (HA) in well water used by the inhabitants for drinking is one of the possible etiological factors for Blackfoot disease (BFD). In this study, the ability of HA to inhibit cell cycle progression and induce apoptosis in cultured smooth muscle cells (SMCs; A7r5) was investigated. Treatment of the SMCs at various HA concentrations (25-200 microg/mL) resulted in sequences of events marked by apoptosis, as shown by loss of cell viability, morphology change, and internucleosomal DNA fragmentation. HA-induced apoptotic cell death that is associated with loss of mitochondrial membrane potential (Delta Psi m), cytochrome c translocation, caspase-3, -8, and -9 activation, poly ADP-ribose polymerase (PARP) degradation, dysregulation of Bcl-2 and Bax, and upregulation of p53 and phospholyrated p53 (p-p53) in SMCs. Flow cytometry analysis demonstrated that HA blocked cell cycle progress in the G1 phase in SMCs. This blockade of cell cycle was associated with reduced amounts of cyclin D1, CDK4, cyclin E, CDK2, and hyperphosphorylated retinoblastoma protein (pRb) in a time-dependent manner. Apparent DNA strand breaks (DNA damage) were also detected in a dose-dependent manner using Single-cell gel electrophoresis assay (comet assay). Furthermore, HA induced dose-dependent elevation of reactive oxygen species (ROS) level in SMCs, and antioxidant vitamin C and Trolox effectively suppressed HA-induced DNA damage and dysregulation of Bcl-2/Bax. Our findings suggest that HA-induced DNA damage, cell cycle arrest, and apoptosis in SMCs may be an underlying mechanisms for the atherosclerosis and thrombosis observed in the BFD endemic region.

Humic acid induced genotoxicity in human peripheral blood lymphocytes using comet and sister chromatid exchange assay.

Humic acid (HA) in well water used by the inhabitants for drinking is one of the possible etiological factors for blackfoot disease (BFD). Moreover, within BFD endemic areas cancers occur at significantly higher rates than in areas free of BFD. In this study, the genotoxic potential of HA is assessed using human peripheral blood lymphocytes. The cells were exposed to HA (0-200 microg/mL for 2 h), and the induction of DNA primary damage in cellular DNA was evaluated by single-cell gel electrophoresis (comet assay). HA-induced DNA damage was decreased by superoxide (O(2)(-)), hydrogen peroxide (H(2)O(2)), and reactive oxygen species (ROS) scavengers (superoxide dismutase, catalase, and Trolox), and nitric oxide (NO) synthase inhibitors (N(G)-nitro-l-arginine methyl ester and N(G)-methyl-l-arginine). Moreover, formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Endo III), known to catalyze the excision of oxidized bases, increase the amount of DNA migration in HA-treated cells. Pretreatment of the cells with both the Ca(2+)-chelator BAPTA and EGTA completely inhibited HA-induced DNA damage, indicating that HA-induced changes in Ca(2+)-homeostasis are the predominant pathways for the HA induction of genotoxicity. Furthermore, sister chromatid exchange was found in the HA-treated lymphocytes. Our findings suggest that HA can induce oxidative DNA damage and genotoxicity in human lymphocytes.

The diabetogenic effects of the combination of humic acid and arsenic: in vitro and in vivo studies.

Black foot disease (BFD) is a peripheral arterial occlusive disease found among the inhabitants of the southwest coast of Taiwan. Moreover, within the BFD-endemic areas, diabetes mellitus occur at significantly higher rates than in other areas of Taiwan. A high concentration of humic acid (HA), and arsenic (As) are present in the artesian well water from BFD-endemic area. The aim of this paper is to study the diabetogenic effect of the combination of HA and AS. Treatment of HIT-T15 cells with HA, As, or both of them resulted loss of cell viability, apoptosis, depletion of ATP, increment of oxidative stress, activation of caspase 3, and dysfunction of insulin secretion. In addition, the plasma insulin of ICR mice, which were exposed to HA and As in drinking water for 12 weeks, was decreased in the 5, 7, and 12 weeks, and increased at early stage of exposure (3 weeks). The results reported herein reveal that HA and As exert HIT-T15 cell dysfunction and inhibited insulin secretive effects. In addition, the sub-acute peri-pancreatitis and islet damage caused by the infiltration of inflammatory cells after exposure of HA and As in drinking water for 5 weeks. Our study has important implications in the diabetogenic effect of the HA and AS which may be mediated by ROS and further information of the toxicity mechanisms will provide under our progressive studies.