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Aquatic Toxicology 105 (2011) 640– 642

Contents lists available at SciVerse ScienceDirect

Aquatic Toxicology

jou rn al h om epa ge: www.elsev ier .com/ locate /aquatox

issolved humic substances initiate DNA-methylation in cladocerans

tefanie Menzela, Rihab Bouchnaka,b, Ralph Menzela, Christian E.W. Steinberga,∗

Humboldt-Universität zu Berlin, Department of Biology, Laboratory of Freshwater & Stress Ecology, Späthstraße 80/81, D-12437 Berlin, GermanyDepartment of Freshwater Conservation of the Brandenburg University of Technology Cottbus, D-15526 Bad Saarow, Germany

r t i c l e i n f o

rticle history:eceived 29 July 2011eceived in revised form 26 August 2011

a b s t r a c t

DNA-methylation is one pathway of epigenetic programming of gene expression and can be responsive toenvironmental challenges such as methylating agents in the food. Here we report on the DNA-methylationin the cladocerans Daphnia magna and Moina macrocopa exposed to humic substances, ubiquitous bio-

ccepted 30 August 2011

eywords:NA-methylationumic substancesaphnia magnaoina macrocopa

geochemicals. The methylation of DNA can alter the stress response, presumably including exposure tosynthetic xenobiotic chemicals.

© 2011 Elsevier B.V. All rights reserved.

Humic substances (HSs) are major biogeochemicals presentn all ecosystems. They are chemically variable and function-lly heterogeneous polymers derived from plant decomposition,ccounting for up to 95% of dissolved organic carbon (DOC) inreshwater bodies; quantitatively, DOC exceeds all carbon in liv-ng organisms by approximately one order of magnitude (Wetzel,001). However, in the perception of whether or not HSs inter-ct with exposed organisms, there seems to be a clear discrepancyetween different scientific disciplines. Whereas medical scholarselate even a severe disease in Taiwan to specific structural prop-rties of consumed HSs (Hseu et al., 2002; Nath et al., 2011; Yangt al., 2004) or discuss various HSs in relation to inflammation,any aquatic toxicologists continue to consider HSs in an indirectode. HSs are thought to be directly inert to aquatic organisms,

xerting only indirect effects through binding of metals and organicollutants (Akkanen and Kukkonen, 2001; De Schamphelaere et al.,004; Haitzer et al., 1999).

Recent studies, however, present evidence that HSs, or at leastome of their building blocks, are taken up by plants and animals.oreover, they transcriptionally interact with biochemical con-

tituents and signaling pathways (Menzel et al., 2005; Trevisant al., 2010). They exert a stress to the exposed organisms, whichauses several stress symtomes, e.g. lipid peroxidation and induc-ion of the biotransformation system. This leads to a multible stress

esistance and can therefore be regarded as beneficial for the organ-sms (Steinberg et al., 2008). More recently, even transgenerationalffects were observed in HS-exposed animals. For instance, Suhett

∗ Corresponding author. Tel.: +49 030 6322 4715; fax: +49 030 6369 446.E-mail address: christian ew steinberg@web.de (C.E.W. Steinberg).

166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.aquatox.2011.08.025

et al. (2011) used life-table experiments to test the effects of nat-ural HSs from a polyhumic Brazilian coastal lagoon and salt on thelife-history of the cladoceran Moina macrocopa Straus. They foundthat pre-exposure to HSs significantly increased body volume ofyoung females in treatments with salt presence, when comparedto non-pre-exposed animals. This indicates that the effects of HSswere even transgenerational. Similarly, exposure of Daphnia magnaStraus females to HSs was an unequivocal stress to which theyresponded by, e.g., production of ephippia. The numbers of ephip-pia can be taken as measure of stress (Bouchnak and Steinberg,2010). Exposed P0 females obviously developed a stress resistancewhich was passed to the F1 and F2 females. In F1 females, noephippia were observed when exposed in the same manner as P0individuals. Interestingly, this stress resistance was less strong inF2 individuals; they produced an intermediate number of ephip-pia (Fig. 1). These HS-impacts on offspring can be based on simpleeffects, where eggs or embryos get direct stress-response infor-mation (proteins, mRNAs) from their mothers, or on epigeneticmechanisms. Vandegehuchte et al. (2009a) recently showed thatD. magna possesses the genetic code for a DNA-methyltransferase.Furthermore, they found that exposure to environmental chemi-cals known to change DNA-methylation in mammals also affectedthe parental generation of D. magna (Vandegehuchte et al., 2010)and that D. magna alters its DNA-methylation status in response toZn-exposure (Vandegehuchte et al., 2009b). In the present study,we tested clones of two cladoceran species in terms of DNA-methylation if exposed to a HS-preparation.

The tested D. magna clone was obtained from the GermanFederal Environment Agency (Berlin). The clone of M. macro-copa originated from a temporary pond in Rio de Janeiro State,Brazil (Elmoor-Loureiro et al., 2010). Both cladocerans were

S. Menzel et al. / Aquatic Toxicology 105 (2011) 640– 642 641

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Fig. 1. Daphnia magna mothers (P0) pass stress resistance to filial generations. Expo-sure for all generations was identical to 20 mg l−1 HuminFeed® (8.6 mg/L DOC).The exposed daughters from exposed mothers (F1) do not produce ephippia ataeS

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Fig. 2. Increased cytosine-methylation in clones of two cladoceran species (Daphnia

ll, whereas exposed granddaughters from exposed daughters (F2) start to producephippia again which are significantly lower in number than their grandmothers. *ignificantly different from P0 at p < 0.05.

ultured in ADaM artificial Daphnia medium (Klüttgen et al., 1994),nder controlled laboratory conditions for more than 3 years prioro exposure. Organisms were cultured in a volume of 10–20 mLer individual for M. macrocopa and D. magna, respectively. AsS-preparation, HuminFeed® was applied at a concentration of5 mg/L DOC. This concentration is environmentally realistic, since. magna is common in ponds, including even stabilization pondsf simple sewage treatment systems and M. macrocopa toleratesuddles impacted by manure (Petrusek, 2002). HuminFeed®1 is

processed leonardite, which is identical to that applied in thetudy of Meinelt et al. (2007) and contains 43% organic carbon, has

specific UV absorption of 12.5 L/mg/m, and consists of 82% HSs,8% low-molecular weight compounds, and 0% polysaccharides.uminFeed® did not change the pH of the culture. Organisms were

ed every second day with the algae Pseudokirchneriella subcapi-ata (6–8 × 105 cells per mL). Gut depuration of cladocerans waserformed by exposing them for 6 h to a suspension of 70 mg/Lne kaolin clay particles (PolsperseTM 10, Imerys, Cornwall, Eng-

and). All exposures were carried out in four independent treatmenteries.

Genomic DNA was extracted from females after their thirdrood in order to gain sufficient amounts of material. D. magnaas 10 and M. macrocopa 7 days old. 20–40 adult organismser replicate for D. magna and M. macrocopa, respectively, wereinsed with deionized water. Water fleas were lysed in 700 �l TENuffer (20 mM Tris/HCl, pH = 7.5; 50 mM EDTA; 100 mM NaCl), 50 �l0% SDS and 10 �l proteinase K (Carl Roth, Karlsruhe, Germany)20 mg/ml), followed by incubation for 2 h at 55 ◦C (constant shak-ng or vortexing every 10 min). After 1 h incubation, another 10 �lf proteinase K was added. Purification of DNA was carried outith phenol/chloroform extraction followed by ethanolic precip-

tation in the presence of 0.3 M sodium acetate. The precipitationook place either at −20 ◦C over night or at −80 ◦C for 30 min. Afterentrifugation for 25 min at 13,000 rpm and 4 ◦C, the pellet wasesuspended in TEN buffer and treated with 10 �g/mL RNase A (Carloth, Karlsruhe, Germany) for 1 h at 37 ◦C. Subsequently, anotherhenol/chloroform extraction was carried out. The precipitatedNA was resuspended in TE buffer (10 mM Tris/HCl, pH = 8.0; 1 mMDTA), the resulting concentration was determined by measuringhe absorption at 260 nm. To evaluate the DNA-methylation status,

he MethylFlashTM Methylated DNA Quantification Kit (Fluoromet-ic) (Epigentek, NY, USA) was used according to the manufacturer’srotocol. In this assay, DNA is bound to strip wells that are

1 The use of HuminFeed® is no advertisement for this product. For morenformation of this product, visit http://www.humintech.com/001/animalfeeds/roducts/huminfeed.html (accessed July, 2011).

magna, Moina macrocopa) exposed to humic substances. The data are means of fourindependent treatments; *p < 0.05.

specifically treated to have a high DNA affinity. The methylatedfraction of DNA (5-methylcytosine) is detected using capture anddetection antibodies and then quantified fluorometrically. Theamount of methylated DNA is proportional to the relative fluores-cence units measured, which can be calculated with the includedformulas for absolute quantification of 5-methylcytosine using astandard curve (Epigentek, NY, USA). The input amount of DNAwas 100 ng. Mean values were calculated for the methyl content ofthe DNA; statistical significance was evaluated by one-way ANOVA(Sigma Stat 3.5, SPSS Inc., USA). Variations were calculated as stan-dard error of the mean.

The results are displayed in Fig. 2. It is obvious that exposureof both cladocerans to HSs leads to an increase of methylated DNAby a factor of 1.54 (Moina) and 1.73 (Daphnia), respectively. Frommedical sciences, it is well understood that the fate of a gene andits product is not only defined by the DNA sequence per se, butalso by the manner how the gene is marked and programmed by,e.g., DNA-methylation. Since the applied HS-preparation containsrelative high quantities of aluminum (Meinelt et al., 2007), thiscontamination could also be responsible for the observed methyla-tion effect. Yet, the only available paper describing Al-effects onDNA-methylation shows that this metal leads to demethylationin tobacco plants (Choi and Sano, 2007). Provided that Al inducesdemethylation also in cladocerans, the gross impact of HSs must beeven stronger in the presence than in the absence of Al in order toreveal the observed significant increases in DNA-methylation. Suchepigenetic programming of gene expression can be responsive toenvironmental challenges mediated by agents in the food (Szyf,2009) and prepare the organism for another challenge to come,for instance synthetic xenobiotics. So, there is increasing evidencethat expression levels of xenobiotics processing genes and nuclearreceptors are regulated by epigenetic mechanisms (Klaassen et al.,2011). To the best of our knowledge, this is the first evidencethat even a ubiquitous biogeochemical matrix, namely HSs, pos-sesses the ability to induce methylation in exposed invertebrates.Although tested in an independent trial, the DNA-methylationinduced by HSs could even be responsible for the transgenerationalstress resistance shown in Fig. 1. This means that an environmen-tal trigger could affect the programming of genes. The advantage tothe organisms would be in facilitating a more rapid response if thestress (including chemical ones) recurs. It would provide the benefitof enhanced protection without the costs associated with constitu-tive expression of stress related genes (Bruce et al., 2007). Overall,

the present results show that environment itself can regulate theresponse of organisms challenged by environmental triggers andthat this response pattern may be passed to succeeding genera-tions. We are just at the beginning of understanding that kind of

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nvironmentally triggered stress regulation in organisms (also, seeandegehuchte and Janssen, 2011).

This work was supported by the German Research FoundationDFG, grant number STE 673/16-1 to R.M.). Furthermore, the Syriantate’s scholarship to R.B. is gratefully acknowledged.

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