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Enrichment of Humic Material with Hydroxybenzene Moieties Intensifies Its Physiological Effects on the Nematode Caenorhabditis elegans Ralph Menzel,*,† Stefanie Menzel,†,‡ Sophie Tiedt,† Georg Kubsch,§ Reinhardt St€oßer,§ Hanno B€ahrs,† Anke Putschew,|| Nadine Saul,† and Christian E. W. Steinberg† †
Department of Biology, Freshwater and Stress Ecology, Humboldt-Universit€at zu Berlin, Sp€athstr. 80/81, 12437 Berlin, Germany Institute of Biochemistry, Freie Universit€at Berlin, Thielallee 63, 14195 Berlin, Germany § Department of Chemistry, Structural Analysis & Environmental Chemistry, Humboldt-Universit€at zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany Department of Water Quality Control, Technical University Berlin, Str. des 17. Juni 135, 10623 Berlin, Germany
)
‡
bS Supporting Information ABSTRACT: Dissolved humic substances are taken up by organisms and interact on various molecular and biochemical levels. In the nematode Caenorhabditis elegans, such material can promote longevity and increase its reproductive capacity; moreover, the worms tend to stay for longer in humic-enriched environments. Here, we tested the hypothesis that the chemical enrichment of humic substances with hydroxybenzene moieties intensifies these physiological effects. Based on the leonardite humic acid HuminFeed (HF), we followed a polycondensation reaction in which this natural humic substance and a dihydroxybenzene (hydroquinone or benzoquinone) served as reaction partners. Several analytical methods showed the formation of the corresponding copolymers. The chemical modification boosted the antioxidant properties of HF both in vitro and in vivo. Humic substances enriched with hydroxybenzene moieties caused a significantly increased tolerance to thermal stress in C. elegans and extended its lifespan. Exposed nematodes showed delayed linear growth and onset of reproduction and a stronger pumping activity of the pharynx. Thus, treated nematodes act younger than they really are. In this feature the modified HF replicated the biological impact of hydroquinone-homopolymers and various plant polyphenol monomers, thereby supporting the hydroxybenzene moieties of humic substances as major effective structures for the physiological effects observed in C. elegans.
’ INTRODUCTION Humic substances (HS) are complex organic molecules that represent the largest constituent of natural organic matter (NOM) in the soil (∼60%);1 they also make up most (50 80%) of the dissolved NOM in freshwater ecosystems.2 4 HS possess a variety of molecular structures and are considered as key components of both soil and freshwater ecosystems. Humic substances are able to bind a variety of organic and inorganic compounds due to their hydrophobic and mainly aromatic structural features, including metal ions via S-, N-, and carboxyl groups; thereby, they modulate the bioavailability of chemical compounds. 5 9 Besides such indirect effects, there is increasing evidence that humic substances can also be directly effective. Thus, humic substances are taken up by organisms, as has often been demonstrated,10 14 and once internalized they can positively influence the metabolic and signaling pathways involved in plant development by directly acting on specific physiological targets such as root formation and nitrogen metabolism.15 17 Humic substances were also found to act as r 2011 American Chemical Society
a mild source of natural chemical stress and as ‘natural biocides’ in relation to a fish-pathogenic bacterium,18 an aquatic macrophyte,19 a fungus,20 and different species of algae;21 hormone-like effects of humic substances have even been described in fish 22 and amphibians.23 In this study, we used the bacteriovorous nematode Caenorhabditis elegans (Maupas, 1900) as the model organism, which belongs to the most abundant class of metazoan organisms in soils and sediments with a high biodiversity. H€oss et al.24 and Steinberg et al. 25 described the impact of different humic substance isolates on the reproductive capacity of this nematode. While the majority of humic substances tested were found to stimulate reproduction in the nematode, a fulvic acid fraction Received: July 8, 2011 Accepted: September 9, 2011 Revised: September 7, 2011 Published: September 09, 2011 8707
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Environmental Science & Technology from a raised peat bog lake decreased reproduction. In a more recent paper, it was demonstrated that C. elegans is gradually attracted by different humic substances;26 moreover, a processed leonardite (HuminFeed, HF) significantly prolonged its lifespan, whereas a polysaccharide-rich NOM isolate of Lake Schwarzer See did not.27 This difference was attributed to the corresponding chemical compositions of the humic substances applied. However, substantial progress in the research on humic substances is still hampered by the lack of a detailed characterization of the material used. Several authors tried to approach this problem with case studies relating the available chemical parameters of various humic substances to biological effects. These data served as the basis of a statistical evaluation to identify potentially effective structures in the sources tested. Overall, the biocide-like impact of specific humic substances, both on the aquatic macrophyte Ceratophyllum demersum19 and on the fish-pathogenic fungus Saprolegnia parasitica,20 could be attributed to humic substances with a higher molecular weight and aromaticity, which probably contain a higher number of stable semiquinone radicals, which may influence the redox homeostasis of an organism. In particular, phenolic and quinonoid moieties have been hypothesized as being highly active.19,20 Indeed, humic substances are redox-active compounds. Evidence has been accumulated to show that they, and particularly their hydroxybenzene moieties, play an important role as electron shuttles in, for example, microbial redox reactions involved in metal reduction and the biodegradation of priority pollutants.28 31 Whether or not these phenolic and quinonoid moieties are also responsible for the rather beneficial impact of humic substances on, for example, the increased reproduction and lifespan of C. elegans is unknown and comprises the central hypothesis of the present study. In this context, it is particularly noticeable that several polyphenolic substances, such as tannic and gallic acid, which are expected to form the building blocks of humic substances,32 are able to significantly extend the lifespan of C. elegans.33,34 In this work, we intended to enrich a selected humic substance with hydroxybenzene moieties via chemical modification rather than perform another correlation analysis using an extensive number of different humic sources. We followed a copolycondensation reaction35 in which natural humic substances and a dihydroxybenzene (hydroquinone or benzoquinone) served as reaction partners in a formaldehyde condensation reaction. Because coalderived humics, mostly consisting of an aromatic core,36 have been considered to be the best candidates for this type of modification,35 we selected HF. Moreover, we added a synthetic, humic-like substance that was synthesized by autoxidation of hydroquinone at a high pH. This humic-like substance consists of aliphatically linked phenolic rings with functional hydroxybenzene groups. The aim of this study was to investigate whether chemically modified or synthetically produced humic substances are able to alter basic physiological parameters in terms of lifespan and thermotolerance, reproductive capacity, pharyngeal pumping activity, and linear growth. Moreover, the property of humic substances as antioxidant to quench free radicals by hydrogen donation and their potential to attract the worms were evaluated.
’ EXPERIMENTAL SECTION Nematodes and Cultivation. The C. elegans wild-type strain Bristol N2 was used throughout this study. The worms were
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maintained on NGM agar37 plates using Escherichia coli OP50 as a food source, according to standard procedures. 38,39 The specified humic substance was added to both the agar and bacterial suspension at concentrations of 0, 0.2, 0.4, and 2.0 mM dissolved organic carbon (DOC). The incubation temperature was always 20 °C. A synchronous culture was generated by rinsing worms from NGM plates and filtering them through a 10 μm membrane (SM 16510/11, Sartorius AG, G€ottingen, Germany), a pore size that retains all but the first-stage juveniles (L1). Humic Substances. Origin. HuminFeed (Humintech GmbH, D€usseldorf, Germany) was made by an alkaline extraction process of highly oxidized lignite. Prior to extraction, the raw lignite material was milled and homogenized. The humate solution was separated from the humin via sedimentation of the latter. Detailed physicochemical analyses in comparison to several other humic sources are available.20 HuminFeed was used for practical reasons and by no means as an advertisement for this product. Chemical Modification. First, formaldehyde polycondensation was conducted between HF and hydroquinone under the conditions taken from Perminova et al.35 HuminFeed (1 g) was dissolved in 2.5 mL of distilled water and further diluted in water to a volume of 50 mL; the pH was adjusted to pH 7. The addition of 500 mg of the monomer hydroquinone (Merck, Darmstadt, Germany) was performed in the presence of catalytic amounts of oxalic acid and 1 g of a 35% solution of formaldehyde. The mixture was stirred for 1 h at 100 °C. The dissolved humic substance was precipitated by the addition of 6 M HCl at pH 1, filtrated, and freeze-dried. This product was called HuminfeedHydroquinone (HF-HQ). For the second modification, 1 g of HF was dissolved in 2.5 mL of distilled water and diluted to 50 mL with 0.1 M KH2PO4 (pH 7). Then, 500 mg of benzoquinone was added, and the mixture was stirred for 1 h at room temperature. Subsequently, the humic substance was precipitated with 6 M HCl at pH 1, filtrated, and freeze-dried. In line with the modification performed, the product was called HF-BQ. Elemental Analysis. Carbon, hydrogen, nitrogen, and sulfur were determined after the combustion of dried samples by a thermal conductivity detector. The metal contents were analyzed by an atomic absorption spectrometer (Perkin-Elmer, MA, USA) after digestion with nitric acid in a high pressure microwave oven (MLS GmbH, Leutkirch im Allg€au, Germany). Synthesis of a Hydroquinone-Homopolymer. Hydroquinone (5 g) was dissolved in 200 mL of 0.2 M Na2CO3 solution and vented with air at 30 L/h for 5 h. The dissolved polymerized hydroquinone was precipitated by the addition of 6 M HCl at pH 1, filtrated, and freeze-dried. This product was called synthetic hydroquinone-homopolymer (sHQ). Size-Exclusion Chromatography (SEC). The size distribution of DOC was determined using size-exclusion chromatography with UV254 detection and online carbon detection. The SEC was carried out using an LC-OCD system (DOC Labor Dr Huber, Karlsruhe, Germany). A Toyopearl HW-50S column was used (250 20 mm, particle size 20 40 μm, Tosoh Bioscience, Tokyo, Japan) for the separation. The compounds were eluted with a 20 mM phosphate buffer (pH 6.5) at a flow rate of 1.5 mL/min. After separation and detection of the UV absorption the mobile phase passed through a Graentzel thin film reactor, where the organic compounds were oxidized after the addition of concentrated phosphoric acid (0.4 mL/min) and irradiation with UV light at 184 nm. The carbon dioxide produced was 8708
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Environmental Science & Technology transferred into an infrared detector with nitrogen 5.0 (20 L/h) as carrier gas. For more details, the reader is referred to Sachse et al.40,41 Furthermore, the specific absorbance of the humic substances was determined. The slope of the absorption curves, as measured by the ratios of absorbance at 465 and 665 nm (E4/ E 6 ratio), has traditionally been suggested to be inversely related to the degree of condensation of aromatic groups (aromaticity).42 Electron Paramagnetic Resonance (EPR)-Spectroscopy. The EPR-spectroscopy provides information on spin concentrations in the material, which are indicative of stable free radicals, which, in turn, can react with many chemical structures, including biotic macromolecules. The Cw X-band EPR-spectra of freeze-dried samples were recorded at room temperature using a spectrometer of the type ERS300 (Centre of Construction of Scientific Devices/Magnettech GmbH, Berlin, Germany) at a microwave power (PMW) of 2 mW, as described in Paul et al.43 Spin concentrations (used to determine the number of unpaired spins per gram of carbon) were determined using strong pitch as a reference. Nuclear Magnetic Resonance (NMR)-Spectroscopy. The 1H MAS NMR spectra were recorded on a Bruker AVANCE 400 spectrometer (Larmor frequency: ν1H = 400.1 MHz) using a 4 mm MAS probe with rotors made from ZrO2. The spectra were recorded with a π/2 pulse duration of p1 = 2.5 μs, a recycle delay of 5 s, and a spinning frequency of 10 kHz. Values of the isotropic chemical shifts are given with respect to tetramethylsilane (TMS). The existing background signals of 1H were suppressed by the application of a phase-cycled depth pulse sequence according to Cory et al.44 Lifespan Assay. This assay accurately followed the protocol published recently.45 In order to start the test, freshly grown L4 larvae (P0) were placed onto either control or treatment plates. Then, L4 larvae of the next generation (F1) were transferred to assay plates with 0, 0.2, 0.4, and 2.0 mM DOC of each humic substance tested. The first day of adulthood was defined as day 1. We performed two independent trials, each comprising ten small agar plates (Ø = 35 mm) with overall 150 nematodes per trial. Alive and dead animals were scored daily until all worms had died. Thermotolerance Test. The F1 generation nematodes were cultured as described above starting from L4 on either control or humic substance-containing plates. During the reproductive period, adult nematodes were transferred daily to fresh treatment plates. On the sixth day of adulthood the agar plates were switched to 35 °C for 7 h; subsequently, the dead and alive nematodes were counted. This thermal resistance test was performed with 100 nematodes for each concentration in three independent trials. Reproduction Assay. Again, L4 larvae of the F1 generation were placed onto individual treatment plates (n = 12 per concentration, two independent trials). Nematodes were transferred daily to fresh treatment plates until reproduction ended, typically day 4 of adulthood. The offspring of each individual nematode was counted once grown to the L2 or L3 stage. Growth Alterations. For the growth assay, 20 nematodes of the F1 generation per concentration and trial (n = 4) were utilized. On the first and sixth day of adulthood, respectively, the nematodes were killed by heat exposure, and the length of each individual nematode was measured using a microscope equipped with a graduated eyepiece. Pharyngeal Pumping Frequency. On the third, sixth, and ninth day of adulthood the pharyngeal pumping frequency was
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quantified. Nematodes of the different treatments were randomly selected (n g 10), and the pumping frequency was determined three times over a 15 s time span. Three independent experimental trials were performed for each assay. Attraction Assay. The NGM agar plates were prepared according to our previously published protocol,26 with six alternating spots of bacteria, lacking or supplemented with humic substances. Total numbers of offspring present after 96 h on the three control spots and the three spots supplemented with humic substances were summed and compared as percentages. Four plates were used for each concentration, and all tests were comprised of three independent experimental trials. Antioxidant Capacity. The superoxide anion radical scavenging activities (SOSA) of both pure humic substances and homogenates of C. elegans were analyzed via photoluminescence for the sum of hydrophilic antioxidants (=ascorbic acid equivalents) in a PHOTOCHEM device and the corresponding ACW-Kit (AnalytikJena, Jena, Germany) according to Popov and Lewin.46 This assay measures the ability of an antioxidant to quench free, photochemically generated superoxide anion radicals by hydrogen donation. The sensitive luminometric detection uses luminol as detector substance. Before measurement, pure humic substances were dissolved in distilled water to a final concentration of 500 μg/mL DOC and neutralized with NaOH to pH = 7 (to provide conditions similar to those used for the biological tests). For worm exposure, about 5000 synchronized L1 larvae were treated with humic substances for three days until they reached the young adult stage. The animals were rinsed from the agar plates with ice-cold M9 buffer, washed three times, and homogenized in a Speedmill (AnalytikJena, Germany). Finally, the resulting homogenate was centrifuged at 2000 rpm for 2 min at 4 °C and immediately used for measurement. The antioxidant capacities in the worms were related to their protein content, which was measured according to Bradford,47 using Bradford reagent (Sigma-Aldrich, MO, USA). All measurements were performed in triplicate. Statistical Analysis. Median and mean lifespan and percentage changes (compared to controls) were determined. The statistical significance of alterations in the mean lifespan was calculated using a log-rank test (Bioinformatics at the Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; http://bioinf.wehi.edu.au/software/russell/logrank). Mean survival rates and percentage changes to the control were calculated for the thermal stress resistance assays. Statistical significance was defined via the chi-square test (SigmaStat 3.5; SPSS Inc., Chicago, IL) and one-way ANOVA (SigmaStat 3.5; SPSS Inc., Chicago, IL) for the daily and total reproductive output, body length, the attraction assay, the pharyngeal pumping frequency, and the antioxidant capacity.
’ RESULTS AND DISCUSSION The objectives of this study were to synthesize and characterize humic substances enriched with hydroxybenzene moieties and to estimate their biological impact on the nematode C. elegans. It was expected that the introduction of additional dihydroxybenzene groups into the structure of the humics would be reflected in changes to the physical and chemical characteristics, for example, spectral and redox properties and, hence, would lead to an enhancement of their beneficial biological properties in exposed nematodes, such as lifespan extension and increased stress resistance. 8709
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Figure 1. The superoxide anion radical scavenging activities (SOSA). (A) Mean total of the antioxidant capacity of four different humic substances, measured in vitro as pure substances and compared to gallic acid and tannic acid (n = 3). Please note the logarithmic scale of the Y-axis. (B) SOSA of water-soluble substances in homogenates obtained from humic substance-treated and untreated wild-type nematodes. The tests were repeated three times with a total of approximately 5000 nematodes per treatment in each single trial. The data are shown as equivalents of nM ascorbic acid and are related to mM dissolved organic carbon (A) or the total protein content (B).
Structural Studies of the Chemically Modified Humic Substances. In their pioneering work, Perminova et al.35 sup-
plied substantial evidence confirming the success of the intended hydroxybenzene-enrichment of leonardite humic substance by using monomeric dihydroxybenzenes; our chemical analyses of modified HF material further validated this protocol. The data are provided in the Supporting Information. A first investigation of the elemental composition and atomic ratios showed a principal similarity between the different data sets (Table S1, Supporting Information). However, the lower H/C atomic ratios of 0.88 (HF-HQ) and 0.79 (HF-BQ) in comparison to 0.95 for the starting material (HF) were the first indicator of the higher aromaticity of the modified humic substances. This was accompanied by a decrease in the E4/E6 ratio, which is inversely related to the degree of aromaticity.42 For HF-HQ and HF-BQ, the E4/E6 ratios were about 20% lower than for the original HF (Table S1, Supporting Information). Further evidence for the successful modification was provided by the increase in spins per gram of carbon, as measured by EPR (Table 1, Supporting Information). The highest spin concentrations, indicative of the content of stable free radicals, were found in HF-BQ and HFHQ. The corresponding single lines gave g-values of g = 2.0031 (or 2.0033 for HF-BQ), which were very similar for all of the samples. They can be attributed to organic radicals and not, for example, to metal ions, which give quite different values.48 The measured g-values (i.e., g∼2.003) are known for humic acids and indicate that the organic radicals were most likely part of the aromatic subsystems, whereas higher g-values (i.e., g close to 2.004) are typical for semiquinone-type radicals.49 The increase in the g-value in the case of HF-BQ provides a further indication of the enrichment of original HF with hydroxybenzene moieties. The results of the SEC (Table S1, Figure S1A, Supporting Information) were very similar for HF and both modifications and comprised the typical main peak which is referred to humic substances as classified by DOC fingerprint analysis of various aquatic samples containing humic substances41 (typically between 3500 and 4000 g/mol). Beside that main signal compounds of lower molecular weight were detected (RT < 60 min, typically 1500 2000 g/mol) and, in case of HF and HF-HQ, higher molecular weight compounds (RT < 45 min, typically 6000 7000 g/mol). Because the higher molecular weight compounds were UV-active at 254 nm (data not shown), we assumed that it did not comprise polysaccharides, which are not detectable by UV.
Our fourth sample, sHQ, a polycondensed hydroquinonehomopolymer, produced a qualitatively similar elution pattern, yet the peaks appeared after prolonged retention times (Figure S1A, Supporting Information). Here, the condensation was done by a different mechanism, and the SEC shows that the polymerization of HQ leads to polymers with a smaller molecular size as found after the reaction of HF with HQ, indicating that the polymerization of HF with HQ was indeed successful. This shift in retention time was exactly reproduced using another polycondensed hydroquinone-homopolymer, HS1500 (Figure S1A, Supporting Information), which was commercially produced by the Sopar Pharma GmbH (Mannheim, Germany). The assumption of a higher aromaticity of the modified humic substances, as discussed above, was supported by the two 1H MAS NMR spectra shown in Figure S1B (Supporting Information). Although both modified (HF-HQ) and unmodified (HF) humic substances gave 1H signals at the same position (Table S1, Supporting Information), their intensities were different. The modified HF-HQ had an unambiguous higher intensity in the region of ring protons or double bonds (7 ppm) (Figure S1B, Supporting Information). Together, all of the data provided the conclusion that the polycondensation of HF and the selected dihydroxybenzenes (HQ and BQ) led to the formation of corresponding copolymers and not simply mixtures of humic substances and hydroquinonehomopolymers. Chemical Modification Boosts the Antioxidant Capacity of HF. Humic substances are known to interfere in redox and radical processes, and their antioxidant capacity can be used to evaluate these properties.50 As a final step in the characterization process, we measured the SOSA of HF before and after the chemical modifications. The relative results are presented and correspond to equivalents of ascorbic acid (Figure 1A). Whereas the original HF only possesses a few nM equivalents per mM DOC, enrichment with dihydroxybenzenes increased this capacity by more than 8-fold (HF-BQ) or even 17-fold in the case of HF-HQ. Hence, the chemically modified HFs showed an antioxidant capacity similar to the hydroquinone-homopolymer sHQ. The quenching capacities even approached those we found for polyphenols such as gallic and tannic acid, which were measured as positive controls (Figure 1A). Both substances were selected because the same and related compounds, in particular those resulting from the enzymatic degradation of lignin, 8710
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Figure 2. Survival curves of C. elegans in the absence or presence of humic substances, which were added at three different concentrations: 0.2 mM (A), 0.4 mM (B), and 2.0 mM DOC (C). Day 1 refers to the first day of adulthood. The statistical significance of alterations in the mean lifespan was calculated using a log-rank test, P < 0.001 (**). Please note that all individual experimental trials of HS exposure, performed independently, produce statistically significant different results in comparison to the control. (D) On the sixth day of adulthood, the number of dead and alive animals was counted following a 7 h exposure to a thermal stress of 35 °C. The columns present the mean values of three independent experimental trials, in each case comprising 5 20 nematodes. The error bars represent the standard error of the mean (SEM), and differences compared to the control were considered significant at P < 0.05 (*) and P < 0.001 (**).
significantly contribute to the antioxidant capacity of humus.51 The strong rise in SOSA as a result of the chemical modification provided further evidence that both HF-HQ and HF-BQ were successfully enriched with hydroxybenzene moieties. In the next step we wanted to evaluate the capacity of modified humic substances to modulate the redox homeostasis of whole organisms (= the potential to balance reactive oxygen species, ROS). In the lower exposure concentrations (0.2 and 0.4 mM DOC), only 0.2 mM HF-BQ-treated nematodes revealed the presence of significantly enhanced antioxidant capacity (Figure 1B). The SOSA of the nematodes treated with the original HF was found to be slightly reduced in comparison to the control (Figure 1B). Only the highest exposure concentration (2 mM DOC) increased the antioxidant capacity in the organisms. The SOSA values of sHQ and both chemically modified HF humics were significantly increased not only in comparison to the control but also to the original HF-derived samples (P between 0.033 and 0.004). Similar results were obtained for several polyphenol monomers, for example, quercetin and caffeic acid52 as well as tannic and ellagic acid,53 which are all well-known to possess substantial antioxidant properties. Why did the in vivo situation not produce an adequate concentration-effect relationship and why did SOSA even decrease in response to the HF treatment (0.2 and 0.4 mM DOC)? As known redox shuttles, humic substances may also exhibit pro-oxidant characteristics depending on the individual redox status of the humic molecule, the inorganic chemistry of the cell, and the quality of nutrients available. These effects interfere with the intrinsic antioxidant capacity of humic substances and can exceed its impact. This may explain several findings published recently, showing for example a decrease in total antioxidant capacity of the blood of Japanese quails treated with humic acid (600 mg/kg food),54 and
higher amounts of oxidatively damaged DNA indicated by an increase in 8-oxodGuo levels in both human primary fibroblasts55 and maternal mice.56 Moreover, leaf litter leachates have been shown to function as pro-oxidants, as determined in an aquatic macrophyte.57 Treatment with Humic Substances Extend the Lifespan in Wild-Type Nematodes. The lifespan of untreated (control) and humic substance-treated wild-type nematodes was compared. Pure HF treatment resulted in a significantly enhanced lifespan at all three concentrations tested (Figure 2A-C), whereas the lower concentrations (0.4 and 0.2 mM DOC, see Figure 2B and A) proved to be the most effective. Almost the same results were obtained for the HF-BQ treatment (Figure 2A-C). In the case of HF-HQ and the synthetic polycondensed sHQ, each concentration tested increased the mean lifespan in comparison to the other humic substances tested. With more than 22% and 17%, the highest gains in mean lifespan were achieved using 0.4 mM DOC of sHQ and HF-HQ, respectively. Both underlying data sets were significantly different in comparison to the original HF data (Log-rank test; P = 0.0054 and 0.0066, respectively). Moreover, by reaching a lifespan extension of about 20%, only these two modifications reached the degree of impact of polyphenol-monomers, for example quercetin45 and tannic acid,34 which extended the lifespan of C. elegans by 18% and 18 20%, respectively. Nematodes Exposed to Modified Humic Substances Exhibit Enhanced Resistance to Thermal Stress. Because longevity has frequently been found to be accompanied by enhanced thermal tolerance, both in long-lived mutant strains58,59 and in response to polyphenols,34,45,52,60 we preferred in this work the thermal stress resistance test over tests following an exposure to reactive oxygen species or oxidizing reagents. The latter assays 8711
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Figure 3. The impact of humic substances on reproduction and growth in length. Total (A) and daily (B) reproductive output was determined from 12 animals per substance and concentration in two independent trials. Day 1 refers to the first day of adulthood. The length of treated and untreated nematodes was measured on both the first (C) and the sixth day (D) of adulthood. In each case, 20 nematodes were used for growth measurements (n = 4). Each bar/point illustrates the average of all associated trials. The error bars represent the standard error of the mean (SEM), and differences compared to the control were considered significant at P < 0.05 (*) and P < 0.001 (**).
often produced contradictory results, as shown for tannic acid,34 rosmarinic acid, and caffeic acid52 as well as an extract of blueberry polyphenols,60 and were not able to enlighten a key effect as longevity is. In order to analyze how humic substances affect the response to thermal stress, nematode survival was monitored following a 7 h exposure to 35 °C. Again, worms treated with humic substances achieved better results than the control animals (Figure 2D). However, only treatment with the chemically modified HFs and sHQ resulted in a significantly enhanced stress tolerance (Figure 2D). Also, HF-HQ and sHQ again caused the highest impact on the nematodes by increasing thermal stress resistance by up to 30% (0.2 mM DOC). So far, humic substances have only been proven to be an inducer of enhanced stress resistance in M. macrocopa, where indigenous humics isolated from coastal lagoons promoted resistance to salt stress.61 Polyphenol monomers, on the other hand, were repeatedly characterized as efficient inducers of stress resistance, in response to both thermal and oxidative stimuli (for C. elegans, see refs 34,45,51, and 52). The finding that only chemically modified humic substances and the hydroquinone-homopolymer sHQ (in the used concentrations) were able to replicate the impact of polyphenol monomers provides a further piece of evidence to show the biological impact of the hydroxybenzene moieties of humics. Exposure to 0.2 mM DOC of HS Slightly Increases Reproduction; Higher Concentrations Reduce Growth in Length. A significant extension of an organism’s lifespan may take place at the expense of reproductive capacity and/or growth.62,63 The lowest concentration of 0.2 mM DOC consistently increased the reproductive capacity of C. elegans for all humic substances tested (Figure 3A). A closer look at the distribution of brood size over the four reproductive days revealed a significant
reduction in the initial reproduction capacity on the second day for the chemically modified HFs and sHQ compared to the control. This reduction, however, was overcompensated on day 3 (HF-BQ and sHQ) and/or day 4 (HF-HQ and sHQ) (Figure 3B). The original HF did not produce this specific effect. When polyphenol monomers were again taken as the reference, tannic, gallic, and ellagic acid were proven to delay reproduction in C. elegans, but they did not modulate the total reproductive capacity.52 However, a set of different humic substances was found to stimulate reproduction in the nematode;24,25 measurements of a potential delay in the onset of reproduction were not performed in previous studies. The size of treated and untreated nematodes was determined on the first (Figure 3C) and sixth (Figure 3D) day of adulthood. On both days, exposure to the highest concentration of humic substances (2.0 mM DOC) resulted in a significant reduction in body length. When the concentration of humics was at 0.4 mM DOC, this effect persisted with HF-HQ, HF-BQ, and sHQ but only on the first day of adulthood. Again, the initial delays were compensated for over time. The increasing content of hydroxybenzene moieties, as mediated by an increased total concentration of humics or by a specific enhancement via chemical modification, seemed to be the trigger. These data again match the findings for the tannic acid52 and catechin polyphenols.64 Indications for an Impact on Eating Behavior. Previous work26 revealed that C. elegans possesses an intrinsic prevalence for humic substances; here, we support this finding. The nematodes were attracted to bacteria spiked with HF-substances or sHQ as indicated by the number of eggs that were laid on the humic-treated bacterial spots (Figure 4A). Differences between the specific treatments became apparent when we compared the concentration-effect relationships. Whereas the preference for 8712
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Figure 4. Humic substances modulate eating behavior. (A) Attraction assay results: the percentage distribution of F1 offspring on agar plates with six alternating spots of bacteria, lacking or supplemented with humic substances, are shown. Open bars refer to the offspring present on the control spots, the filled bars correspond to offspring present on the spots supplemented with humic substances. (B) The pumping activity of 3 10 (three independent trials) treated and untreated nematodes was monitored on the third, sixth, and ninth day of adulthood (from left to right). Each individual nematode was quantified three times for 15 s. The error bars represent the standard error of the mean (SEM), and differences compared to the control were considered significant at P < 0.05 (*) and P < 0.001 (**).
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pumping frequency on all days, whereas the lower concentration (0.2 mM DOC) as well as the higher concentration (2.0 mM DOC) significantly increased pharynx activity only on the sixth and ninth day of adulthood (Figure 4B). Obviously, the older the nematodes are, the more they can profit from treatment with humic substances. Chemical intervention to modify the original HF did not further enforce the activating property of HF on the pumping frequency of C. elegans. Several polyphenols also share this property with humic substances. Quercetin, rosmarinic, and caffeic acid45 as well as gallic acid and catechin,52 were found to mediate an increase in pharyngeal activity. As with the humics, the relative gain in activity upon polyphenolic exposure peaked in each case on the ninth day of adulthood. It is well-known that the pumping frequency of C. elegans reaches its maximum in the young adult stage, followed by a continuous decrease until death.65 Taking the observed delays in reproduction and growth in length into account, the higher pharyngeal activity reflected a phenotype where the nematodes seemed to look younger than they really were. The enhancement of HF with hydroxybenzene moieties intensified this effect, also seen by the increased thermotolerance and the extended lifespan. In conclusion, chemical modification of humic substances from a leonardite source, in order to enhance its hydroxybenzene moieties, resulted in a gain of beneficial properties. Almost all of the variables measured responded to the modifications and the observed values resembled the findings obtained for the hydroquinone-homopolymer sHQ and polyphenol monomers. The major effect seems to be a delay in aging. Our data support that the hydroxybenzene moieties of humic substances were mainly responsible for the physiological effects observed in the nematode C. elegans.
’ ASSOCIATED CONTENT
bS
Supporting Information. Please find here chemical parameters (Table S1) as well as DOC-fingerprint analyses and 1H MAS NMR spectra (Figure S1) of the humic substances. This material is available free of charge via the Internet at http:// pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
the original HF showed its highest measured value at 2.0 mM DOC, the concentration dropped to 0.4 mM in the case of HFHQ and HF-BQ. Furthermore, sHQ attracted most of the nematodes at the lowest concentration of 0.2 mM DOC, whereas 2.0 mM DOC was not effective. With the polyphenol monomers, the preferential behavior of C. elegans was heterogeneous. Whereas tannic and gallic acid were able to attract the nematodes,52 quercetin, rosmarinic, and caffeic acid did not,45 and ellagic acid even repelled C. elegans.52 Our data suggest that the chemical modifications of HF (or the polymerization of hydroquinone to sHQ) enhanced the fraction of moieties in the HS mediating the attracting properties; a process which led to a decline of the most efficient DOC concentration. The observed preference for humic substances at certain concentrations was also reflected by an increase in pharynx activity, as determined by counting the pumping frequency on the third, sixth, and ninth day of adulthood (Figure 4B). All humic substances tested significantly increased the pumping frequency. The concentration of 0.4 mM DOC increased the
*Phone: +4930 63224241. Fax: +4930 6369446. E-mail: ralph.menzel@ biologie.hu-berlin.de.
’ ACKNOWLEDGMENT This work was supported by the grant STE 673/16-1 awarded by the ‘Deutsche Forschungsgemeinschaft’ (DFG). We are grateful to Dr. Gudrun Scholz (HU Berlin, Institute for Chemistry) for excellent support and generation of parts of the NMR figure. ’ REFERENCES (1) Stevenson, F. J. Organic forms of soil nitrogen. In Humus Chemistry: Genesis, Composition, Reactions; Stevenson, F. J., Ed.; John Wiley & Sons: New York, 1994; pp 59 95. (2) Thurman, E. M. Aquatic Humic Substances. Organic Geochemistry of Natural Waters; Nijhoff-Junk: Dordrecht, 1985. (3) Steinberg, C. E. W.; M€unster, U. Geochemistry and ecological role of humic substances in lakewater. In Humic Substances in Soil, 8713
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