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PubMed2002

Inhibition of endogenous thyroid hormone receptor-beta and peroxisome proliferator-activated receptor-alpha activities by humic acid in a human-derived liver cell line.

Humic acid (HA), know to be ubiquitous in the natural environment, is present in almost all soil, surface water, and plants. Earlier studies indicate that HA can affect thyroid economy via binding with iodide, inhibiting both thyroid peroxidase and hepatic 5'-deiodinase in rodents. However, the effect of HA, a peroxisome proliferator in rodents, on thyroid hormone receptor (TR) and peroxisome proliferator-activated receptor (PPAR) in human cells has not yet been examined. In this study, we demonstrate that the malic enzyme activity and the transcriptional activities of endogenous TR and PPAR were inhibited after treatment with HA in human hepatocyte Chang liver cell line. Although the protein expression levels of TR-beta, PPAR-alpha and retinoid X receptor-alpha (RXRalpha) were not changed significantly by HA treatment, both the binding abilities of endogenous TR-beta on thyroid hormone response element (TRE) and PPAR-alpha on the PPAR response element (PPRE) were inhibited by HA treatment. The study of the subcellular distribution of HA, relying on the inherent HA fluorescence, showed that HA distributed in the intracellular compartments including cytoplasm and nucleus. The 50% binding inhibition values (CI(50)) of HA on ME-TRE (malic enzyme gene-TRE) and ACOX-PPRE (acylCoA oxidase gene-PPRE) were 19.31 and 19.94 microg/mL, respectively. These results suggest that HA-induced endemic goiter may link in part to the disruption of TRbeta and PPARalpha function in human Chang liver cells. This model may be useful in the investigation of environmental goitrogens.

Blotting, Western↗
PubMed1996

In vivo cytogenetic effects of natural humic acid.

As humic compounds are naturally widespread in the environment and present in surface water, studies on their genotoxicity are justified. Humic acid (HA) has not been demonstrated to be genotoxic either in vitro or in vivo. In the present paper we investigated its activity both in intestinal and bone marrow cells following a single dose (100 mg/kg b.w. corresponding to 0.5 ml per animal of an aqueous solution of 4 g/l) of HA administered to mice by gastric intubation, to mimic the most likely route of human exposure. HA induced structural and, in particular, numerical chromosome abnormalities in intestinal cells. A marginal, non-significant induction of aneuploidy was also found in bone marrow cells.

Aneuploidy↗
PubMed1998

Efficient removal of PCR inhibitors using agarose-embedded DNA preparations.

The use of agarose blocks containing embedded DNA improves the PCR amplification from templates naturally contaminated with polysaccharides or humic acids, two powerful PCR inhibitors. Presumably, the difference in size between the DNA macromolecules and these contaminants allows their effective removal from the agarose blocks by diffusion during the washing steps, whereas genomic DNA remains trapped within them. In addition, agarose-embedded DNA can be directly used for PCR since low melting point agarose does not interfere with the reaction. This simple and inexpensive method is also convenient for genomic DNAs extracted by other procedures, and it is potentially useful for samples containing other kinds of soluble inhibitors, overcoming this important problem of current amplification techniques.

DNA↗
PubMed2003

Mechanisms underlying the impact of humic acids on DNA quantification by SYBR Green I and consequences for the analysis of soils and aquatic sediments.

DNA quantification of soils and sediments is useful for the investigation of microbial communities and for the acquisition of their genomes that are exploited for the production of natural products. However, in such samples DNA quantification is impaired by humic acids (HA). Due to its lack of specificity and sensitivity, UV spectrophotometry cannot be applied. Consequently, fluorimetric assays applying Hoechst (H) 33258 or PicoGreen (PG) are used. Here, we investigated the SYBR Green I (SG) assay, which was also affected by HA, but was found to be 25- and 1.7-fold more sensitive compared to the H 33258 and PG assays, respectively. Spectrophotometric, fluorimetric and quenching studies as well as gel mobility shift assays suggested that the effect of HA on the SG assay was based on an inner filter effect, collisional quenching and binding of SG to HA. As to the latter finding, the standard 6250-fold dilution of the SG reagent was optimised to a 2000-fold dilution. Although the sensitivity of the optimised SG assay was reduced by a factor of 1.3, the interfering effect of HA could be reduced up to 22-fold. A significant reduction of HA interferences by lowering the pH of the assay was not observed. Finally, the performance of the modified SG assay and the corresponding evaluation methods were verified by the determination of DNA recoveries and concentrations of standards and environmental samples in comparison to the PG assay.

Benzothiazoles↗
PubMed2002

The effects of dietary humate supplementation on broiler growth and carcass yield.

The growth-promoting effect of Farmagülatör DRY Humate (FH) on live performance, carcass weight, and the abdominal fat pad of broilers was studied during different feeding periods. Four hundred, 1-d-old straight-run birds were randomly distributed to 20 floor pens of an environmentally controlled house. Four dietary regimens were replicated in five pens, each containing 20 chicks, as follows: 1) birds received no added FH in the starter or grower (NAFH), 2) birds received FH from 0 to 21 d (FH0-21), 3) birds received FH from 22 to 42 d (FH22-42), 4) birds received FH from 0 to 42 d (FH0-42) in the starter and grower diets, respectively. The FH was added to the diets at 2.5 kg/per ton of feed. Starter and grower diets were formulated to meet the minimum NRC requirements for broilers and were provided as a mash feed. Body weights at 21 d were not affected by the dietary regimens. At 42 d, body weights and feed conversions of broilers were significantly affected by the dietary humate treatments. Birds fed FH22-42 weighed more than the NAFH, whereas the FH0-21 and FH0-42 were intermediate and not different from the other treatments. Feed:gain was lower for the FH22-42 and FH0-42 treatments compared to the NAFH. There was no difference in carcass yield or abdominal fat pad percentages due to feeding FH. Feeding FH during the grower period had the most beneficial effect in terms of growth and feed conversion on broiler performance.

Adipose Tissue↗
PubMed2004

The effects of supplementation of humate and probiotic on egg production and quality parameters during the late laying period in hens.

This study was designed to investigate whether inclusions of humate and probiotic into diets of hens during the late laying period increases egg production and improves egg quality. Hisex Brown layers (n = 300), 54 wk of age, were fed a control diet, 0.1% humate, 0.2% humate, 0.1% probiotic, or 0.2% probiotic for 75 d. Active ingredients of humate and probiotic were polymeric polyhydroxy acids (humic, fulvic, ulmic, and humatomelanic acids) and bacterial cultures (Lactobacillus, Bifidobacterium, Streptococcus, and Enterococcus spp.), respectively. Egg production and feed intake were measured daily, and egg weight was measured biweekly. Also, a sample of 12 eggs from each group was collected randomly to determine egg quality every 25 d. The data were analyzed as repeated measures with time as subplot. There were no effects of dietary treatments on feed intake and egg weight. Egg production for hens supplemented with humate and probiotic was not different but was greater than for control hens. Egg production increased linearly and mortality and feed conversion efficiency (weight of feed/weight of eggs) decreased linearly with increasing levels of supplemental humate and probiotic. There were no effects of treatments on egg quality. In conclusion, supplementation of humate and probiotic during the late laying period increased egg production, reduced mortality, and improved feed conversion efficiency but did not improve egg quality.

Animal Nutritional Physiological Phenomena↗
PubMed2006

Effects of humic acid on broiler chickens.

In view of the alleged effect of humic acid (HA) on growth plate arthrosis in humans, we sought to find if poultry tibial dyschondroplasia (TD) is caused by HA that can occur as a ground water contaminant. In 2 separate trials, broiler chickens were fed different concentrations of HA added to their diet for 4 and 5 wk. The effects of these treatments were measured by general health indices such as growth, feed conversion ratio, relative organ weights, blood differential count, serum chemistry, TD index, and bone biomechanical strength. Humic acid treatment decreased BW but appeared to improve feed conversion ratio. There was no effect on TD index or bone biomechanical strength in HA-treated birds compared with controls. There was no toxic effect of HA that was evident by the absence of any dramatic change in relative organ weights or other telltale signs of serum clinical chemistry that would suggest liver, muscle, or kidney dysfunction. Red blood cell, white blood cell, monocyte, and hematocrit values were not affected, but there was a decrease in blood heterophil counts and heterophil to lymphocyte ratio, which was significant in 4-wk HA-treated birds. Overall these results show that HA slows down growth, but it does not have any adverse health effects on chickens.

Animals↗
PubMed2006

In vitro and in vivo assessment of humic acid as an aflatoxin binder in broiler chickens.

The in vitro affinity and adsorption capacity of a humic acid, oxihumate, for aflatoxin B1 (AFB1) was evaluated, utilizing Langmuir and Freundlich adsorption isotherms. Oxihumate showed a high in vitro affinity for AFB1. The Freundlich isotherm fitted the data better than the Langmuir isotherm, and binding capacities of 10.3, 7.4, and 11.9 mg of AFB1/g of oxihumate at pH 3, 5, and 7, respectively, were calculated. The in vivo efficacy of oxihumate as an aflatoxin binder in male broiler chickens exposed to aflatoxin-contaminated feed from 7 to 42 d of age was also assessed. The efficacy of oxihumate was compared with a commercially available product with a brewers dried yeast (BDY) and brewers fermentation solubles as main active ingredients. A total of 420 birds were assigned to 28 pens, with 15 birds per pen. The following treatments were applied: 1) 0 mg of AFB1 + 0 additives, 2) 1 mg of AFB1/kg of feed + 0 additives, 3) 1 mg of AFB1/kg of feed + 3.5 g of oxihumate/kg of feed, 4) 1 mg of AFB1/kg of feed + 3.5 g of BDY/kg of feed, 5) 2 mg of AFB1/kg of feed + 0 additives, 6) 2 mg of AFB1/kg of feed + 3.5 g of oxihumate/kg of feed, and 7) 2 mg of AFB1/kg of feed + 3.5 g of BDY/kg of feed. Each treatment consisted of 4 replicates. Oxihumate was effective in diminishing the adverse effects caused by aflatoxin on BW of broilers (P < 0.05). Oxihumate also showed protective effects against liver damage, stomach and heart enlargement, as well as some of the hematological and serum biochemical changes associated with aflatoxin toxicity (P < 0.05). Results indicated that oxihumate, but not BDY, could alleviate some of the toxic effects of aflatoxin in growing broilers. Oxihumate might, therefore, prove to be beneficial in the management of aflatoxin-contaminated feedstuffs for poultry when used in combination with other mycotoxin management practices. Additional studies are warranted to assess its efficacy under a wide variety of circumstances.

Adsorption↗
PubMed1995

Potential effects of surface water components on actinide determinations conducted by ion chromatography.

An elution program for separating actinides (thorium, uranium, neptunium, plutonium, americium, and curium) on low hydrophobicity ion exchange columns was evaluated for solutions spiked with actinides and common surface water components. Potential interferences from dissolved ions (Na+, K+, Ca2+, Cl-, and SO(4)2-), humic acid, and radium were investigated. Sulfate levels greater than 0.25 mumol interfered with separation of americium, curium, and plutonium. Humic acid levels above 100 micrograms produced distinct widening of actinide peaks and reduced actinide recoveries. These interferences limit the range of useful sample volumes and create a need for sample pretreatment procedures. No interferences were produced by 0.025 to 2.5 mumol Ca2+, 0.045 to 4.5 mumol Na+, 0.015 to 1.5 mumol K+, and 0.025 to 4.5 mumol Cl-. In the absence of interferences, the program effectively separated radium from the actinides.

Actinoid Series Elements↗
PubMed2002

Potential trace metal-organic complexation in the atmosphere.

It is possible that metal-organic complexation enhances the uptake of gaseous organic compounds and the solubility of metals in aerosols and atmospheric water. We investigated potential atmospheric organic ligands and the enhanced uptake of hydroxy-, oxo-, and dicarboxylic acids as well as dicarbonyls into atmospheric aqueous aerosol. We examined complexation with transition metals (iron, manganese, nickel, copper, zinc) and lead on the basis of available references and our experimental data. Humic-like substances are most likely ligands in the atmosphere, although this is a poorly characterized material. A number of polycarboxylic acids and hydroxy forms (e.g., citric and tartronic acids) effectively complex metals such as copper in atmospheric aerosols. The simple equilibrium model calculations show that the effect of the complexation on the gas-aqueous phase partition of gaseous atmospheric ligands is quite small for the ligands with the high physical Henry's law constants, e.g., dicarboxylic acids represented by oxalic acid, even if they have high affinity with metal ions. The lower Henry's law constants of the alpha-dicarbonyls, such as glyoxal and methylglyoxal, mean that the complexation could lead to profound increases in their partition into the aqueous phase. Despite quantum mechanical arguments for copper-glyoxal complexes, experiments showed no evidence of complexation between either hydrated or unhydrated alpha-dicarbonyls and the cupric ion. By contrast the beta-dicarbonyl, malondialdehyde, has properties that would allow it to partition into atmospheric water via the complexation with metal ions under some conditions.

Aerosols↗
PubMed2004

Towards sustainable use of potassium in pineapple waste.

Due to the 1997/98 haze problem in South-East Asia and the increasing need for sustainable food production and development, the usual management of crop residues (including pineapple wastes) through burning is prohibited. As a result, the need for alternative uses of pineapple wastes in pineapple production has been emphasized. This study investigated an environmentally friendly means of recycling pineapple leaves for agricultural use. Pineapple leaves were shredded and composted in a composting drum for 30 days. Part of the shredded leaves was ashed in a muffle furnace for 4 h. Humic acid (HA), K-fulvate, and K in HA and compost were analyzed using standard procedures. An ash to water ratio of 1:7 was used to extract 0.1 molar (M) KOH from the shredded leaves. The 0.1 M KOH contained 50% K and was able to extract 20% HA from the composted pineapple leaves. Percent K in the fulvate using 0.1 M KOH was 43. Besides serving as a foliar spray (supplement soil application K fertilizers), source of K for freshwater fish (e.g., tilapia), the HA produced can be used as a soil conditioner. Studies show that between 0.05-0.01 g of HA per kg soil retards runoff by 36% in sandy and sandy loam soils. The K-fulvate can be used as a fluid fertilizer. In addition, the pH of 2 of the K-fulvate suggests it could be used to dissolve phosphate rocks, particularly those in the arid regions where high soil pH does not facilitate the dissolution of these important rocks that serve as one of the sources of phosphorus fertilizer in agriculture.

Ananas↗
PubMed1991

Sunlight and the survival of enteric bacteria in natural waters.

Escherichia coli and some salmonellas were exposed in seawater and freshwater to natural sunlight, visible light of comparable intensity, and light containing a similar proportion of u.v. as natural sunlight but of a much lower intensity. Direct viable bacterial counts and culturable counts on selective and non-selective media were made at intervals. The rate of decrease in numbers of culturable bacteria was significantly faster in seawater than in freshwater when exposed to natural sunlight. No significant difference was found between the rates of decrease in numbers of culturable bacteria in seawater and those in freshwater when bacteria were exposed to light with a small u.v. component of similar intensity. The effect of salinity no loss of culturability is, therefore, more significant in the presence of u.v. radiation. Direct counts by the acridine orange direct viable count method decreased much more slowly than the culturable counts in seawater but comparably with culturable counts in freshwater in natural sunlight. Direct viable counts and culturable counts decreased at a similar rate in seawater and in freshwater in visible light. This may signify the evolution of enteric bacteria towards a viable but non-culturable form in seawater when exposed to natural sunlight. The presence of humic acids significantly reduced loss of culturability but only in low salinity conditions. Salinity appears to be an important factor influencing culturability in bacteria exposed to sunlight.

Escherichia coli↗
PubMed2005

Microbiological parameters as indicators of compost maturity.

AIMS: The objectives of this study were to determine the changes of microbial properties of pig manure collected from pens with different management strategies and composted using different turning and moisture regimes; relate their association with humification parameters and compost temperature; and identify the most suitable microbial indicators of compost maturity. METHODS AND RESULTS: Six different microbial parameters, including total bacterial count, oxygen consumption rate, ATP content, dehydrogenase activity, and microbial biomass C and N, along with humification parameters [humic acid (HA), fulvic acid (FA) and HA : FA ratio] and compost temperature were monitored during composting. Significant positive correlations were found between temperature and microbial properties, including O2 consumption rate, ATP content, dehydrogenase activity, and microbial biomass N. The humification parameters also showed significant correlations with microbial properties of the manure compost. For instance, HA contents of pig manures was positively correlated with total aerobic heterotrophs, and microbial biomass N and C; and negatively correlated with O2 consumption rate, ATP content, and dehydrogenase activity. Among the six microbial parameters examined, dehydrogenase activity was the most important factor affecting compost temperature and humification parameters. Composting strategies employed in this study affected the speed of composting and time of maturation. If the moisture content is maintained weekly at 60% with a 4-day turning frequency, the pig manure will reach maturity in 56 days. CONCLUSIONS: The composting process went through predictable changes in temperature, microbial properties and chemical components despite differences in the initial pig manure and composting strategies used. Among the six microbial parameters used, dehydrogenase activity is the most suitable indicator of compost maturity. Compared with respiration rate, ATP content and microbial biomass procedures, dehydrogenase activity is the simplest, quickest, and cheapest method that can be used to monitor the stability and maturity of composts. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented here show that microbial parameters can be used in revealing differences between composts and compost maturity. The statistical relationship established between humification parameters and microbial parameters, particularly dehydrogenase activity, demonstrates that it is possible to monitor the composting process more easily and rapidly by avoiding longer and more expensive analytical procedures.

Adenosine Triphosphate↗
PubMed2006

Effect of iron(III), humic acids and anthraquinone-2,6-disulfonate on biodegradation of cyclic nitramines by Clostridium sp. EDB2.

AIMS: To determine the biodegradation of cyclic nitramines by an anaerobic marine bacterium, Clostridium sp. EDB2, in the presence of Fe(III), humic acids (HA) and anthraquinone-2,6-disulfonate (AQDS). METHODS AND RESULTS: An obligate anaerobic bacterium, Clostridium sp. EDB2, degraded RDX and HMX, and produced similar product distribution including nitrite, methylenedinitramine, nitrous oxide, ammonium, formaldehyde, formic acid and carbon dioxide. Carbon (C) and nitrogen (N) mass balance for RDX products were 87% and 82%, respectively, and for HMX were 88% and 74%, respectively. Bacterial growth and biodegradation of RDX and HMX were stimulated in the presence of Fe(III), HA and AQDS suggesting that strain EDB2 utilized Fe(III), HA and AQDS as redox mediators to transfer electrons to cyclic nitramines. CONCLUSIONS: Strain EDB2 demonstrated a multidimensional approach to degrade RDX and HMX: first, direct degradation of the chemicals; second, indirect degradation by reducing Fe(III) to produce reactive-Fe(II); third, indirect degradation by reducing HA and AQDS which act as electron shuttles to transfer electrons to the cyclic nitramines. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study could be helpful in determining the fate of cyclic nitramine energetic chemicals in the environments rich in Fe(III) and HA.

Anthraquinones↗
PubMed2007

Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils.

AIM: To establish a rapid, improved soil environmental DNA extraction and purification protocol. METHODS AND RESULTS: Three different soil DNA isolation and four purification strategies were compared on different soil samples with variable rates of success. Bead beating extraction gave significantly higher DNA yields than microwave-based and liquid nitrogen grinding DNA extraction methods. The inclusion of soil washing prior to cell lysis decreased the amount of purification steps required. Although these soil types differed, polyvinylpolypyrrolidone (PVPP)-sepharose 2B column elution was sufficient for all three samples, yielding DNA pure enough for successful application in molecular studies. One soil sample retained 80% of the initial DNA after successful purification. CONCLUSIONS: Optimization of a purification protocol confirmed that only a combination of previously described methods proved sufficient in yielding pure environmental DNA from humic-rich soils. Total processing time for DNA extraction and subsequent purification from multiple samples was considerably more rapid than the previously described methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a new optimized soil DNA extraction and purification protocol that is suitable for different environmental sources that are rich in humic acid content.

Bacillus↗
PubMed2006

Microbial community dynamics in a humic lake: differential persistence of common freshwater phylotypes.

In an effort to better understand the factors contributing to patterns in freshwater bacterioplankton community composition and diversity, we coupled automated ribosomal intergenic spacer analysis (ARISA) to analysis of 16S ribosomal RNA (rRNA) gene sequences to follow the persistence patterns of 46 individual phylotypes over 3 years in Crystal Bog Lake. Additionally, we sought to identify linkages between the observed phylotype variations and known chemical and biological drivers. Sequencing of 16S rRNA genes obtained from the water column indicated the presence of phylotypes associated with the Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, TM7 and Verrucomicrobia phyla, as well as phylotypes with unknown affiliation. Employment of the 16S rRNA gene/ARISA method revealed that specific phylotypes varied independently of the entire bacterial community dynamics. Actinobacteria, which were present on greater than 95% of sampling dates, did not share the large temporal variability of the other identified phyla. Examination of phylotype relative abundance patterns (inferred using ARISA fragment relative fluorescence) revealed a strong correlation between the dominant phytoplankton succession and the relative abundance patterns of the majority of individual phylotypes. Further analysis revealed covariation among unique phylotypes, which formed several distinct bacterial assemblages correlated with particular phytoplankton communities. These data indicate the existence of unique persistence patterns for different common freshwater phylotypes, which may be linked to the presence of dominant phytoplankton species.

Bacteria↗
PubMed2007

217 000-year-old DNA sequences of green sulfur bacteria in Mediterranean sapropels and their implications for the reconstruction of the paleoenvironment.

Deep-sea sediments of the eastern Mediterranean harbour a series of dark, organic carbon-rich layers, so-called sapropels. Within these layers, the carotenoid isorenieratene was detected. Since it is specific for the obligately anaerobic phototrophic green sulfur bacteria, the presence of isorenieratene may suggest that extended water column anoxia occurred in the ancient Mediterranean Sea during periods of sapropel formation. Only three carotenoids (isorenieratene, beta-isorenieratene and chlorobactene) are typical for green sulfur bacteria and thus do not permit to differentiate between the approximately 80 known phylotypes. In order to reconstruct the paleoecological conditions in more detail, we searched for fossil 16S rRNA gene sequences of green sulfur bacteria employing ancient DNA methodology. 540 bp-long fossil sequences could indeed be amplified from up to 217 000-year-old sapropels. In addition, such sequences were also recovered from carbon-lean intermediate sediment layers deposited during times of an entirely oxic water column. Unexpectedly, however, all the recovered 16S rRNA gene sequences grouped with freshwater or brackish, rather than truly marine, types of green sulfur bacteria. It is therefore feasible that the molecular remains of green sulfur bacteria originated from populations which thrived in adjacent freshwater or estuarine coastal environments rather than from an indigenous pelagic population.

Benzopyrans↗