Modulating effects of humic acids on genotoxicity induced by water disinfectants in Cyprinus carpio

The use of chlorinated disinfectants during drinking-water production has been shown to generate halogenated compounds as a result of interactions of humic acids with chlorine. Such chlorinated by-products have been shown to induce genotoxic effects and consumption of chlorinated drinking-water has been correlated with increased risk for cancer induction in human populations. The aim of this work was to test the potential genotoxic effects on circulating erythrocytes of the fish Cyprinus carpio exposed in vivo to well-waters disinfected with sodium hypochlorite (NaClO), chlorine dioxide (ClO2) or peracetic acid (CH3COO2H, PAA), in the absence or presence of standard humic acids (HA). The effects were measured by use of the micronucleus (MN) and the single-cell gel electrophoresis (Comet) assays at different sampling times after a 3-day exposure period. The exposure to chlorine disinfectants without the addition of HA produced a clear toxic effect. Significant cytogenetic damage (i.e. MN induction) was detected in fish populations exposed to both NaClO and ClO2 with humic acids. In the Comet assay, a significant decrease of DNA migration was observed in erythrocytes of specimens after exposure to NaClO-disinfected water without HA. No effects were observed in any other experimental condition.     

Degradation of humic acids by the litter-decomposing basidiomycete Collybia dryophila

The basidiomycete Collybia dryophila K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic (14)C-labeled humic acid ((14)C-HA) prepared from [U-(14)C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn(2+) (200 microM), leading to 75% conversion of LHA and 50% mineralization of (14)C-HA (compared to 60% and 20%, respectively, in the absence of Mn(2+)). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn(2+)-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation, C. dryophila released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as C. dryophila which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.     

Mechanism of the desmutagenic effect of humic acid

The mechanism of an apparent desmutagenic effect of humic acid was investigated. Firstly, components of humic acid (resorcinol, vanillin, vanillic acid, ferulic acid, protochatechuic acid and benzoic acid) were tested and were not found to show a desmutagenic effect. By contrast, lignin did show a desmutagenic effect. The desmutagenic effect of humic acid was decreased by ozone treatment, and the degree of decrease corresponded with a decrease in KMnO4 consumption. Benzo[a]pyrene and humic acid were incubated at 37 degrees C for 1 h and extracted by ethyl acetate and the extract was investigated by gas chromatography (GC). The peak of the decomposition product did not appear, but the amount of benzo[a]pyrene was decreased. This suggests that the desmutagenic effect of humic acid was caused by adsorption of benzo[a]pyrene by humic acid rather than by decomposition of benzo[a]pyrene. Humic acid had the largest adsorption activity at its critical micelle concentration (CMC), while adsorbed benzo[a]pyrene could be released by ultrasonication. Fulvic acid and water-soluble humic substance showed a slight inhibitory effect on the mutagenicity of benzo[a]pyrene.     

Ellagic acid protects rat embryos in culture from the embryotoxic effects of N-methyl-N-nitrosourea.

Ellagic acid is a naturally occurring plant phenol that has demonstrated anticarcinogenic and antimutagenic activity in several test systems. Given the common proposed etiopathogenic processes of mutagenesis, carcinogenesis, and teratogenesis induced by genotoxic chemicals, the present study was initiated to determine whether ellagic acid would protect rat embryos in culture from the teratogenic effects of N-methyl-N-nitrosourea (MNU). Ellagic acid alone (as used in these experiments; 50 microM in DMSO) was not embryotoxic. Ellagic acid (50 microM) significantly (P less than 0.01) prevented MNU (75 microM)-induced effects including mortality (absence of heart beat), abnormal formation of the cephalic neural tube derivatives, and delayed differentiation as assessed by a morphological scoring system. These embryoprotective effects were dose responsive. Sequential treatment of embryos with ellagic acid followed by MNU in fresh media also was embryoprotective with no diminution of effect. The site at which ellagic acid interrupts the critical teratogenic events induced by MNU is apparently within the embryo and/or placenta. This model of chemical embryoprotection may be useful in determining the role of cell death and/or mutation in the teratogenic mechanism of action of methylating agents.     

Effects of municipal waste compost and sewage sludge on proton binding behavior of humic acids from Portuguese sandy and clay loam soils

The effects of amendment with municipal solid waste compost (MSWC) and sewage sludge (SS) on acid-base properties of soil humic acids (HAs) were investigated. For this purpose, HAs were isolated from MSWC and SS and two different Portuguese soils, one sandy and the other clay loam, either unamended or amended with MSWC or SS at a rate of 60 t ha(-1), and analysed by potentiometric titrations at various ionic strengths (0.01, 0.05, 0.1 and 0.3M) over the pH range from 3.5 to 10.5. All titration data were fitted with the NICA-Donnan model and the variations of model parameters between the various HA samples were discussed. The HAs from MSWC and SS had lower acidic functional group contents and higher proton binding affinities than the control soil HAs. Amending soils with MSWC and SS determined a decrease of acidic functional group contents and an increase on proton binding affinities of soil HAs. These effects were more evident in SS-amended soil HAs than in MSWC-amended soil HAs, and in clay loam soil HA than in sandy soil HA.     

Investigation of genotoxic and antigenotoxic activities of chlorophylls and chlorophyllin in cultured V79 cells

Chlorophyll and its derivatives are examples of plant compounds (purified and/or extracted) which appear to protect DNA from damage caused by chemical or physical agents, although some studies have identified clastogenic activity of these compounds. This study was carried out to assess the genotoxic activity of chlorophyll-a (Chl-a), -b (Chl-b) and chlorophyllin (Chl) and their antigenotoxic activity against the DNA damage induced by methyl methanesulphonate (MMS) under conditions of simultaneous, pre-, post-treatment, and simultaneous treatment after pre-incubation of the chemical with MMS. The micronucleus (MN) test was used in binucleated cells (induced by cytochalasin-B) of a mammalian cell line (V79). The three concentrations of Chl-a, Chl-b or Chl (0.1375, 0.275, 0.55microM) were not genotoxic and the genotoxic action of MMS (400microM) decreased (74-117%) under all treatment conditions. The results showed that there was no significant difference among the treatment types, the concentration or the nature of chlorophyll used. The data obtained suggest that Chl-a, Chl-b and Chl when associated with the DNA damaging agent, MMS, may protect the DNA by desgenotoxic action and/or by bio-antigenotoxic mechanisms, with the similar efficiency.     

Biochemical Origin and Refractory Properties of Humic Acid Extracted from the Maize Plant

Humic acids (HA) contribute to soil fertility because of their chemical, physical, and biological properties. The origin of HAs in soils has puzzled scientists for decades, and what HAs are and what their origin is remain unclear. The isolation of HAs in plants, which have characteristics close to soil HAs, suggests the probable origin of soil-HA is the preservation of plant tissue, indicating biochemical origin. In this paper HA from maize plant at different stages of maturity is isolated, from which it was found that the evolution of this fraction depends on and is derived from cell wall formation. Evidence was also found that HA was above all composed of lignin and cutin residues, and was characterized by low surface area. After 8 months of incubation in both mineral-artificial and natural soils, humic acid isolated form maize plant could be recovered intact.     

Antigenotoxic effect of lipoic acid against mitomycin-C in human lymphocyte cultures

Antitumor agents are used in therapy against many forms of human cancer. One of these is mitomycin-C (MMC). As with many agents, it can interact with biological molecules and can induce genetic hazards in non-tumor cells. One of the possible approaches to protect DNA from this damage is to supply antioxidants that can remove free radicals produced by antitumor agents. Lipoic acid (LA) is known as one of the most powerful antioxidants. The aim of this study was to investigate antigenotoxic effects of LA against MMC induced chromosomal aberrations (CA), sister chromatid exchanges (SCE) and micronucleus (MN) formation in human lymphocytes. Lymphocytes were treated with 0.2 μg MMC/heparinized mL for 48 h. Three different concentrations (0.5, 1, 2 μg/mL) of LA were used together with MMC in three different applications; 1 h pre-treatment, simultaneous treatment and 1 h post-treatment. A negative, a positive and a solvent control were also included. In all the cultures treated with MMC + LA, the frequency of abnormal cells and CA/cell significantly decreased compared to MMC. Statistically significant reduction was also observed in SCE/cell and MN frequencies in all treatments. These results demonstrated anticlastogenic and antimutagenic effects of LA against MMC induced genotoxicity. LA showed the most efficient effect during 1 h pretreatment. On the other hand, MMC + LA treatments induced significant reduction in mitotic index than that of MMC treatment alone. These results are encouraging that LA can be a possible chemopreventive agent in tumorigenesis in both cancer patients and in health care persons handling anti-cancer drugs.     

Effects of sewage sludge amendment on the properties of two Brazilian oxisols and their humic acids

The effect of sewage sludge (SS) amendment on the general properties of the top layers of a sandy and a clayey oxisols and on the nature of their humic acid (HA) fractions was evaluated by chemical and physico-chemical techniques. The amended soils, especially the sandy soil, benefited of SS amendment by increasing their pH to above neutrality and enhancing the contents of C, N, P, and Ca and cation exchange capacity. The SS-HA-like sample showed larger H and N contents and a greater aliphatic character and humification degree than the HAs isolated from non-amended soils. The composition and structure of amended soil HAs were affected by SS application as a function of soil type and layer. In particular, N-containing groups and aliphatic structures of SS-HA-like sample appears to be partially incorporated in the amended soil HAs, and these effects were more evident in the HAs from the sandy oxisol.     

Earthworms as modifiers of the structure and biological activity of humic acids

Passage of humic acids (HAs) through the digestive tract of the earthworm, Eisenia fetida andrei, resulted in a decrease in molecular masses of the HAs. The effect of earthworm-modified HAs on individual bacteria and on bacterial communities as a whole is different from the effect of native HAs. Modified HA probably induces and regulates microbial successions in soils and composts in a different manner than the native HA, suppressing or stimulating different groups of microorganisms. These results may explain why the positive effects of commercial humates in real soil ecosystems, unlike model communities, attenuate rapidly.     

Biochemical origin and refractory properties of humic acid extracted from maize plants: the contribution of lignin

The soil organic carbon (SOC) pool is the largest terrestrial reservoir of carbon and plant residues play an important role in its maintenance. Up to 70–80% of SOC in arable soil is composed of humic substances (HS). In these soils post-harvested residues, left in arable soil after harvesting the crops, are the basic source of humus. Previous research indicated that maize plants residue contain a humic acid (HA) fraction possessing recalcitrant compounds that contributed to soil-HA fraction. This study presents updated results obtained using Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS) to provide an indication of the contribution of the lignin to the soil HA. Results obtained indicated that the HAs from maize plants were mainly composed of lignin-derived moieties that were likely derived from the partial hydrolysis of p-coumaric and ferulic acid that are linked to lignin, polysaccharides or other biopolymers of the cell wall. Lignin composing the HAs derived from plants and incubated in soil was substantially preserved. Nevertheless the modification of the syringyl/guaiacyl ratio and the oxidation of the side-chains of lignin, suggested a turnover of lignin-derived molecules in soil-HA fraction. This fact indicated an involvement of the alkali insoluble fraction of maize plant residue (humin) in the soil-HA formation, up-dating our previous knowledge.     

Desmutagenic effect of humic acid

In the present study, humic acid was not found to be mutagenic and did not inhibit spontaneous mutation. It did, however, inhibit the mutagenicities of benzo[a]pyrene and 3-aminoanthracene (+S9 mix), but not the mutagenicities of 4NQO, AF-2 and MNNG (-S9 mix). 2-Nitrofluorene and 1-nitropyrene do not need S9 mix for activation of their mutagenicities, but an inhibitory effect was observed. Humic acid exerts a desmutagenic effect on mutagens directly before they act on cells. It does not act as an antimutagen which blocks the processes changing normal cells to mutants. The desmutagenic effect was not decreased by heat treatment (120 degrees C, 15 min). Humic acid was fractionated according to molecular weight and the desmutagenic effect increased with an increase in molecular weight. This effect in the fraction with molecular weight exceeding 300 000 was decreased by centrifugation. The desmutagenic ability of humic acid may result from soluble components and adsorption to small particles.     

Induction of an adaptive response in human blood lymphocytes exposed to radiofrequency fields: influence of the universal mobile telecommunication system (UMTS) signal and the specific absorption rate

The induction of an adaptive response (AR) was examined in human peripheral blood lymphocytes exposed to non-ionizing radiofrequency fields (RF). Cells from nine healthy human volunteers were stimulated for 24h with phytohaemagglutinin and then exposed for 20h to an adaptive dose (AD) of a 1950MHz RF UMTS (universal mobile telecommunication system) signal used for mobile communications, at different specific absorption rates (SAR) of 1.25, 0.6, 0.3, and 0.15W/kg. This was followed by treatment of the cells at 48h with a challenge dose (CD) of 100ng/ml mitomycin C (MMC). Lymphocytes were collected at the end of the 72h total culture period. The cytokinesis-block method was used to record the frequency of micronuclei (MN) as genotoxicity end-point. When lymphocytes from six donors were pre-exposed to RF at 0.3W/kg SAR and then treated with MMC, these cells showed a significant reduction in the frequency of MN, compared with the cells treated with MMC alone; this result is indicative of induction of AR. The results from our earlier study indicated that lymphocytes that were stimulated for 24h, exposed for 20h to a 900MHz RF GSM (global system for mobile communication) signal at 1.25W/kg SAR and then treated with 100ng/ml MMC, also exhibited AR. These overall data suggest that the induction of AR depends on RF frequency, type of the signal and SAR. Further characterization of RF-induced AR is in progress.     

Antimutagenic Properties of Bacteria: Review

Lactic acid and propionic acid bacteria, bifidobacteria, and fecal enterococci associated with the activity of humans and animals caused antimutagenic effects (AME) on many test systems designed for detecting point mutations and chromosomal aberrations. Bacterial cells and some of their metabolites attenuate the mutagenic action of several genotoxic agents, and this effect is mediated by the mechanism of dysmutagenesis and/or bioantimutagenesis. Possible mechanisms of various AMEs and possible practical applications of antimutagenic properties of bacteria are discussed.     

Modification of humic acids by the compost-dwelling deuteromycete Paecilomyces inflatus

The soil mold Paecilomyces inflatus is capable of modifying and partially mineralizing synthetic and natural humic acids (HAs) in compost environments. HA degradation studies using a synthetic HA (¹⁴C-HA) in autoclaved compost microcosms showed that, after 12 weeks of cultivation, P. inflatus mineralized approximately 5% of the ¹⁴C-labeled HA to¹⁴CO₂, while 6% of the ¹⁴C-HA was converted into ¹⁴C-labeled water-soluble fragments (fulvic-acid-like fraction). About 40% was still present as NaOH-soluble HA representing unmodified or only slightly modified humic material (compared with 60% in the controls). Modification of natural HAs extracted from compost was followed by their partial decolorization (30%) in liquid cultures of P. inflatus. Bleaching of the medium was accompanied by moderate changes in the molecular mass distribution of both the HA and fulvic-acid fractions, which were analyzed with high-performance size exclusion chromatography. HA modification was most pronounced during the primary growth phase of the fungus and was associated with increased laccase activity.     

Antimutagenic properties of bacteria: a review

Lactic acid and propionic acid bacteria, bifidobacteria, and fecal enterococci associated with the activity of humans and animals caused antimutagenic effects (AME) on many test systems designed for detecting point mutations and chromosomal aberrations. Bacterial cells and some of their metabolites attenuate the mutagenic action of several genotoxic agents, and this effect is mediated by the mechanism of dysmutagenesis and/or bioantimutagenesis. Possible mechanisms of various AMEs and possible practical applications of antimutagenic properties of bacteria are discussed.     

Evaluation of the transformation of organic matter to humic substances in compost by coupling sec-page

Humic acids (HAs) from soil and compost at the beginning (S0) and at the end of the stabilization process after 130 days (S130) have been fractionated by coupling size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE). Preparative quantities of HA fractions (HAFs) with different molecular sizes (MSs) and exactly defined electrophoretic mobility (EMs) have been obtained from all samples and the HAFs weight content has been studied. A high degree of similarity in HAFs weight content between soil HA and a stabilized compost HAs130 has been observed. Such data seem to be reliable for monitoring the evolution of the compost organic matter to humic substances for their agricultural uses.     

Assessment of in vitro bioactivity of hyaluronic acid and sulfated hyaluronic acid functionalized electroactive polymer

Electrically conductive polypyrrole (PPY) was surface functionalized with hyaluronic acid (HA) and sulfated hyaluronic acid (SHA) to improve its surface biocompatibility. The immobilization of HA on the PPY film was facilitated by the use of a cross-linker having the appropriate functional groups. The biological activity of the HA functionalized PPY film was assessed by means of an in vitro PC12 cell culture. The cell attachment on different substrates was studied and determined by bicinchoninic acid protein analysis. Cell attachment on the HA functionalized PPY film surface was significantly enhanced in the presence of nerve growth factor. The SHA functionalized PPY film was obtained by the sulfonation of the immobilized HA using pyridinesulfonate. The retention of the biological activity of the immobilized HA after sulfonation was evaluated by the in vitro assessment of the plasma recalcification time (PRT) and platelet adhesion on the substrate. The PRT observed from the SHA functionalized PPY film was significantly prolonged compared with the HA functionalized PPY. Some reduction of platelet adhesion was observed for the SHA functionalized PPY film, compared with that of the HA functionalized PPY film.     

Oxidative injury induced by synthetic humic acid polymer and monomer in cultured rabbit articular chondrocytes.

Humic substance has been proposed as one of the causative factors of Kashin-Beck disease (KBD), an endemic osteoarthritic disorder with necrosis of chondrocytes widely prevalent in some regions of China. In order to exclude the complications of natural humic substance, here we prepared phenolic polymers of synthetic humic acid (SHA) by oxidation of phenolic monomer, the protocatechuic acid (PCA). The biological effects of SHA and PCA on primary culture of rabbit articular chondrocytes were investigated. We found that not only SHA but also PCA caused chondrocyte injury, as evidenced by the loss of cell viability measured with methylthiazol tetrazolium (MTT) assay and the increased release of intracellular lactate dehydrogenase (LDH). Both SHA and PCA could result in lipid peroxidation and glutathione (GSH) depletion in chondrocytes, indicating that oxidative stress may be involved in chondrocyte injury. Furthermore, a marked increase in intracellular calcium level ([Ca2+]i) occurred after chondrocytes treated with SHA or PCA. These results suggest that chondrocyte injury elicited by SHA or PCA may be mediated through the occurrence of oxidative stress and the disruption of intracellular Ca2+ homeostasis. Data also suggest that the monomeric phenolic acid may be considered one of the causative factors of KBD in addition to humic substance.     

The mutagenic and antimutagenic effects of Ecballium elaterium fruit juice in human peripheral lymphocytes

The aim of this study was to investigate the mutagenic and antimutagenic effects of Ecballium elaterium (EE) fruit juice, which has an anti-inflammatory effect, using in vitro human peripheral lymphocytes. To investigate the mutagenic effects of the EE fruit juice, human peripheral lymphocytes were treated with three doses (18, 36, and 72 μl/l) of fruit juice alone for 24 and 48 h. For investigating the antimutagenic effects of the EE fruit juice, the human lymphocytes were also treated with the mixture of the fruit juice and 0.25 μg/ml MMC. The EE fruit juice induced the percentage of total CA when used alone (especially the percentage of structural CA than the percentage of the numerical CA) and synergically induced the percentage of total CA when used as a mixture with MMC. The EE fruit juice did not affect the SCE frequency for 24 and 48 h treatment time. In contrast, EE and MMC as a mixture sinergically induced the SCE frequency at the highest concentration for 48 h treatment time only. EE alone did not decrease the RI while it decreased the MI in a dose-dependent manner. EE and MMC as a mixture have a higher cytotoxic effect than the cytotoxic effects of EE alone. As a result, it can be concluded that EE had no antimutagenic effect while EE had a mutagenic and a cytotoxic effect in human peripheral lymphocytes. This article was submitted by the authors in English.