Transcriptome Profiling of Wheat Seedlings following Treatment with Ultrahigh Diluted Arsenic...

Research ArticleTranscriptome Profiling of Wheat Seedlings followingTreatment with Ultrahigh Diluted Arsenic Trioxide

Ilaria Marotti1 Lucietta Betti1 Valeria Bregola1 Sara Bosi1 Grazia Trebbi1

Giovanni Borghini2 Daniele Nani3 and Giovanni Dinelli1

1 Department of Agricultural Sciences University of Bologna Viale Fanin 44 40127 Bologna Italy2 Department of Public Health and Infectious Diseases ldquoLa Sapienzardquo University of Rome Piazzale Aldo Moro 5 00185 Rome Italy3 Italian National Health System Lombardy District ldquoAzienda Sanitaria Localerdquo Milan Corso Italia 19 20122 Milan Italy

Correspondence should be addressed to Ilaria Marotti ilariamarottiuniboit

Received 7 August 2014 Revised 29 October 2014 Accepted 30 October 2014 Published 27 November 2014

Academic Editor Kuttulebbai N S Sirajudeen

Copyright copy 2014 Ilaria Marotti et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Plant systems are useful research tools to address basic questions in homeopathy as they make it possible to overcome someof the drawbacks encountered in clinical trials (placebo effect ethical issues duration of the experiment and high costs) Theobjective of the present study was to test the hypothesis whether 7-day-old wheat seedlings grown from seeds either poisoned witha sublethal dose of As

2O3or unpoisoned showed different significant gene expression profiles after the application of ultrahigh

diluted As2O3(beyond Avogadrorsquos limit) compared to water (control) The results provided evidence for a strong gene modulating

effect of ultrahigh diluted As2O3in seedlings grown from poisoned seeds a massive reduction of gene expression levels to values

comparable to those of the control groupwas observed for several functional classes of genes A plausible hypothesis is that ultrahighdiluted As

2O3treatment induced a reequilibration of those genes that were upregulated during the oxidative stress by bringing the

expression levels closer to the basal levels normally occurring in the control plants

1 Introduction

Over the past decades the growth of public interest andpopular use of complementary and alternative medicine(CAM) in the treatment of various health problems have beenaccompanied by an increased number of research articles andevidence-based approaches assessing efficacy and therapeuticeffects Homeopathy one of themost widespread and contro-versial forms of CAM uses medicines obtained from naturalsubstances (minerals plants and animals) subjected to aprocess of serial dilutions (in different scales) that results inextremely low oftennonmeasurable levels of active principle

The central aspect of the dispute concerning the homeo-pathic treatments is that at very high dilution levels (beyondthe Avogadro limit) the probability of the presence ofmolecules of the original substance is near to zero [1 2]which according to conventional scientific thinking makeany biological activity highly unlikely Despite the fact thatthere is extensive evidence for ultrahigh dilution (UHD)

efficacy both in vitro [3ndash6] and in vivo [7ndash12] the cellular andmolecular mechanisms underlying the regulatory processesaffected by UHDs must yet be largely elucidated Variouslines of investigation suggest that UHDs might affect somesubtle and early levels of signal transduction andor geneticexpression [2] However the demonstration of their efficacyin a scientific manner and the exploration of their modeof action within the domain of science have become achallenging field for researchers having to address the manyunanswered questions about UHDs by rigorously applyingthe scientific principles in high quality basic and clinicalresearch [13ndash15]

One of the most accredited hypotheses to explain themechanism of action of UHDs in vivo [16] is based on theregulation of expression of some specific and relevant genesThe modulatory effects of UHDs have been demonstratedin both prokaryotes and a primitive form of eukaryote likeyeast [17 18] According to this hypothesis homeopathicremedies carry specific ldquosignalsrdquo or ldquomolecular imprintsrdquo

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 851263 15 pageshttpdxdoiorg1011552014851263

2 Evidence-Based Complementary and Alternative Medicine

of drug that can be identified by specific receptors of thecells as a trigger to turn ldquoonrdquo or ldquooffrdquo some relevant genesinitiating a cascade of gene actions to alter and correct thegene expressions that might have gone wrong during produc-tion of the pathological disorderdisease condition [16 19]Considering that eukaryotic gene expression is an intricateprocess still not completely understood it is difficult withthe knowledge we have at the moment to precisely suggestthe actual molecularmechanisms involved in transmission ofinformation of homeopathic remedies down to the executionlevel for active recovery process [19] For validating thebasis of UHD efficacy and for understanding their molecularmechanism of action further development of basic researchis highly desirable

The microarray technology has been proven to be asuitable high throughput tool to simultaneously analyze alarge number of genestargets associated with the therapeuticeffects of CAM remedies such as herbal drugs [20] Theworking principle for this genomic approach is that thephenotype of a cell including the function and responseto the environment is ultimately determined by its geneexpression profiles Application of such expression analysistools may help to understand and characterize the genes andsignaling pathways involved in CAMmodalities

Microarray analyses have been used to investigate geneexpression variation in human cell lines and animal modelsafter treatment with high dilutions (HDs) and UHDs [21 22]or homeopathic formulations [23] Recently Saha et al [24]used global microarray profiling of genes to demonstrate thatthe expression of certain genes in cancer cells treated withUHDs was significantly different from that of the placebotreated cells

At present microarray profiling of genes to verify theeffects of UHDs on plant systems has never been usedPlant models appear to be useful approaches to address basicquestions on homeopathy research as they make it possibleto overcome some of the drawbacks encountered in clinicaltrials such as placebo effect ethical issues duration and highcosts [25ndash27] The in vitro growth of wheat coleoptiles is oneof the first models adopted to investigate the effects of UHDs[28] and has become a classic test system for basic research inthis field [29] In previous experiments [30 31] we observed acurative effect of arsenic trioxide (As

2O3) at the 45th decimal

dilutiondynamization level on wheat seedling growth Thistreatment applied after having poisoned the seeds with a sub-lethal dose of As

2O3 induced a significant increase in shoot

length compared to poisoned seeds grown in distilled waterIn the present study we reproduced our original exper-

iment [30] in order to investigate the global effects ofultrahigh diluted As

2O3treatments on wheat coleoptiles

gene expression through microarray analyses (Gene ChipsAffymetrix CA) The overall goal was to test the hypothesiswhether wheat coleoptiles grown from As

2O3poisoned

seeds showed different significant gene expression profilesafter the application of ultrahigh diluted As

2O3compared

to water (placebo) At the present state of our knowledgethis is the first time that the microarray profiling approachis being used on plant organisms for gene expression stud-ies on UHDs This might throw additional insight into

the molecular mechanism involved in the biological action ofultrahigh diluted homeopathic drugs

2 Materials and Methods

21 Plant Material and Experimental Treatments Seeds ofthe common wheat (Triticum aestivum L) cultivar ldquoPandasrdquo(CGS sementi Italy) were selected for integrity uniform sizeshape and colour and used to carry out the study

Part of the wheat seeds was pretreated (poisoned) byimmersion for 30min in a 5mM As

2O3watery solution

(As2O3 puriss pa 9995ndash10005 Sigma-Aldrich St Louis

MO USA H2O pa Merck Darmstadt Germany) rinsed in

tap water for 60min dried at ambient air until 12 humiditywas attained and stored in the darkness until use Thissublethal poisoning protocol was selected on the basis ofprevious range and time exposure-finding trials [30] Controlseedswere pretreated in the sameway but with distilledwaterinstead of As

2O35mM

Each seed (poisoned and unpoisoned) was fixed on theupper part of a filter paper (Perfecte 2-extrarapid CordenonsPordenone Italy) by a piece of clay (020 plusmn 005 g) Thefilter paper was inserted into a transparent polyethylenebag (12 times 20 cm) which in turn was placed into a largerblack cardboard envelope covering the roots but not theshoots [30]This technique ensured the growth of shoots androots in natural light and darkness respectively 32mL ofdistilled water or As

2O345x (freshly prepared and supplied

by Laboratoires Boiron Sainte-Foy-les-Lyon France) wasadded in each polyethylene bag during seed growth [3031] As

2O345x treatment (containing a theoretical dose of

As2O31 times 10minus47M) was prepared according to the European

Pharmacopoeia through serial dilutions (in distilled water)at decimal scale (indicated as x) starting from a mother tinc-ture (As

2O3001M) and intercalated by mechanical shaking

(dynamization process) The distilled water used for controland serial dilutions was from the same batch The followingexperimental groups were set up (1) control (denoted by C +0) seeds grown in distilled water without prior treatment (2)treated control (denoted by C + T) seeds grown in As

2O3

45x without prior treatment (3) poisoned (denoted by P +0) seeds pretreated with a sublethal dose of As

2O3and

subsequently grown in distilled water (4) poisoned-treated(denoted by P + T) seeds pretreated with a sublethal doseof As2O3and subsequently grown in As

2O345x A total of

80 cardboard envelopes (20 per each of the 4 experimentalgroups) were fixed to a wooden support and seeds weregrown in the laboratory at a temperature of 20 plusmn 1∘C indaytime assuring a natural day-night rhythm for 7 daysTreatments (water or As

2O345x) were letter-coded (blind

protocol) by a person not involved in the experimentationand were poured into the polyethylene bags according to acompletely randomized design Seven independent replicatedexperiments were carried out for a total of 140 seedlingsgrown for each group out of 560 processed seedlings

The rationale behind poisoning seeds with arsenic andthen growing seedlings in ultrahigh diluted arsenic comesfrom one of the basic tenets of complementary medicinesusing UHDs the principle of ldquolike cures likerdquo In other words

Evidence-Based Complementary and Alternative Medicine 3

symptoms that are caused by a substance (say As2O3) can be

successfully removed by the application of the same substanceat highly diluted microdoses (say As

2O345x)

In our ldquowheat modelrdquo symptoms of illness were inducedby treating wheat seeds with a solution of arsenic triox-ide Arsenic is a toxic metalloid widely disseminated inthe environment and causes a variety of health and envi-ronmental hazards Due to its widespread occurrence inthe environment every organism from human to bacteriahas mechanism for arsenic detoxification In plant-basedresearch arsenic is commonly used for inducing oxidativestress in studies investigating the response of antioxidantdefense systems The right dosage of As

2O3for the stress

was defined in our previous experimentation [30] in whichwe tested different arsenic aqueous solutions (from 1mM to10mM) for different exposure times (from 30 to 120minutes)The combination arsenic dilution-exposure time reducingseed germination rate by approximately 15 was chosen assublethal treatment for imparting a moderate but measurablestress at the system (As

2O35mM for 30 minutes)

Ultrahigh diluted As2O3is generally used in complemen-

tary alternative medicine for curing symptoms of arsenicpoisoning in patients We transferred this clinical practice toour plant system and ultrahigh diluted As

2O3was provided

to arsenic intoxicated seeds as treatment for the recoveryfrom the oxidative stress Particularly the UHD As

2O345x

(containing a theoretical dose of As2O31 times 10minus47M and

thus beyond Avogadrorsquos limit) was chosen on the basis ofour previous findings showing reproducible and significantstimulating effects on in vitro wheat growth [30 31]

22 RNA Extraction and Microarray Expression ProfilingAmong the seven experimental replications three of themwere chosen for sampling materials for microarray analysesFrom each replicate 10 seedlings were randomly collectedwithin each set (control treated control poisoned andpoisoned-treated) and pooled A total of 3 bulks (biologicalreplicates) per set were obtained and used for RNA extractionand microarray analyses (3 biological replicates per set for atotal of 12 hybridizations)

Total RNA extraction was performed on frozen (minus80∘C)material with the Nucleospin RNA plant kit (MachereyNagel) according to manufacturerrsquos instructions RNA con-centration was determined spectrophotometrically RNAquality controls cRNA preparation labeling hybridizationwashing staining and scanning procedures were performedby Genopolis (Department of Biotechnology and BioscienceUniversity of Milano-Bicocca Milan Italy) as described inthe Affymetrix technical manual Briefly total RNA integritywas assessed with Agilent Bioanalyzer (Agilent 2100 RNA6000 Nano LabChip Agilent Technologies Palo Alto CAUSA) and the RNA integrity number (RIN) was calculatedOnly high quality RNA preparations with RIN greater than7 were used for microarray analysis

Fivemicrograms of total RNA for each samplewas used ina reverse transcription reaction (One-Cycle Target LabelingAssay Affymetrix) to generate first-strand cDNA Aftersecond-strand synthesis double-strand cDNAwas used in anin vitro transcription reaction to generate biotinylated cRNA

After purification and fragmentation and relative qualitycontrol checks by an Agilent Bioanalyzer the biotinylatedcRNA was used for hybridization Fragmented cRNAs werehybridized to the standard arrays for 16 h at 45∘C the arrayswere then washed and stained using the fluidics station andscanned using a GeneChip Scanner 3000 with AffymetrixGCOS software

Total RNA was processed for use on GeneChip WheatGenome Array (Affymetrix Santa Clara CA USA) contain-ing 61127 probe sets which interrogates 55052 transcriptsfor all 21 wheat chromosomes in the genome 59356 probessets represent modern hexaploid (A B and D genomes)bread wheat (T aestivum) and are derived from the publiccontent of the T aestivum UniGene Build 38 (April 242004) 1215 probe sets are derived from ESTs of a diploidnear relative of the A genome (T monococcum) a further 539represent ESTs of the tetraploid (A and B genomes) durumwheat species T turgidum and five are from ESTs of a diploidnear relative of the D genome known as Aegilops tauschiiProbe sets consisted of pairs of 11 perfect match (PM) andmismatch (MM) 25-mer oligonucleotides designed from the31015840 end of exemplar sequences with nucleotide 13 as the MMArray annotation information is available on theNetAffx dataanalysis center httpwwwaffymetrixcom

23 Affymetrix Chip Data Analysis Microarray data areavailable in the ArrayExpress database (httpwwwebiacukarrayexpress) under accession number E-MEXP-3666

Microarray data analysis was performed with the Affym-etrix package encoded in the R language Affymetrix CELfiles were imported using the methods in the MicroarraySuite 50 (MAS5 the standard Affymetrix algorithm) whichinvolves background correction and normalization across allof the chips in the experiment To reduce the number ofnoninformative genes a filter based on an interquartile rangewas applied (interquantile range IQR = 02)

Gene expression profiles were normalized by dividing thevalue for a gene for the control value and then calculatingthe log2 of the ratio The identification of differentiallyexpressed genes (DEGs) was addressed using a linear model-ing approach (Limma) and empirical Bayesian methods [32]together with false discovery rate correction of the 119875 value(adjusted 119875 value le005) [33]The DEGs were further filteredby selecting genes showing a log2 fold change (log2 FC) gt1 orlt minus1

Annotation analysis of induced and repressed genes wasperformed using WheatPLEX a plant ontology database(httpwwwplexdborg) and HarvESTWheatChip (httpharvestucredu) Putative functions were assigned if the 119864-value was less than 1119890minus10 Alignments with a higher scorewere visually inspected and annotated if a reasonable degreeof homology was observed Genes were classed as unknownif no reasonable alignments were found

Pathway analysis was conducted with MapMan 351(httpmapmangabipdorgwebguestmapman-version-351)[34] using the wheat-specific Taes AFFY 0709 mapping fileTranscripts were mapped into a custom MapMan pathway[35] The transcript abundance of each gene was scaled to


Table 1 List of the 55 genes selected for microarray validation by qRT-PCR grouped on the basis of their involvement in stress-relatedfunctional categories

Functional categories Gene IDLipid metabolism BG909536 BJ270709 CK198840 CK207205 CK208220Histones and cellularstructural proteins

BQ838979 CA635455 CA660860 BE416616 BJ251672 BQ607338 CA628396 CD868502 CD869995CA719316 BJ225202 BJ262694 BJ274465 BJ306445 BJ308450 BJ320258 BQ294672 BQ168973

Stress proteins BJ322693 CA613417 CA699507 CA708152 CA708660 CK205521Cell signalingtransduction and transportproteins

BF484849 BJ226504 BJ246114 BJ303391 BJ312841 BQ578355 CA629836 CA640553 CA681335CA744534 CA747224 CD453390 CK203324

Secondary metabolism CK210556Protein metabolism BJ228692 BJ243882 BJ256146 CA599873 CA700404 CA731980Cell detoxification systems CA642191 CA676257 CA676553 CA685752 CA719198 CK198851

range between minus3 and 3 and values were averaged acrosseach BIN

All individual genes within a functional category wererepresented as a square box and their expression levels wereshown in a colour (red-blue) scale

24 Gene Expression Analysis Using Quantitative Real-TimePCR (qRT-PCR) The reliability of the Affymetrix GeneChipdata was confirmed by quantitative RT-PCR (qRT-PCR)analysis of a set of 55 genes chosen on the basis of theirinvolvement in stress-related functional categories (lipidmetabolism histones and cellular structural proteins stressproteins cell signaling transduction and transport pro-teins secondary metabolism protein metabolism and celldetoxification systems) (Table 1) qRT-PCR experiments wereperformed using StepOnePlus (Applied Biosystems UK)RNA fromC+ 0 C + T P + 0 and P + T groups from all threebiological replications used in the GeneChip experiment wasused for validation Reverse transcription was performedusing High-Capacity cDNA Reverse Transcription Kitsac-cording to the standard protocol of the supplier (AppliedBiosystems UK)

A pair of primers for each of the selected genes (Table 2)was designed by following a set of stringent criteria as gen-erally suggested in qRT-PCR protocols (eg Primer ExpressSoftware v20 Application Manual Applied Biosystems)Target sequences were obtained from the Affymetrix websiteEach PCR reaction contained 1x Power SYBR(R) Green PCRMaster Mix (Applied Biosystems UK) 20 ng of cDNA and900 nM of forward and reverse primers in a total volume of20120583L Reaction conditions were as follows 95∘C for 10min40 cycles of 95∘C for 15 s and 60∘C 1min To confirm thespecificity of amplificationmelting curve analysiswas carriedout after the last cycle of each amplification All samples wereamplified in triplicate from the same RNA preparation andthe mean value was considered For each primer pair no-template controls were also run Quantification of the relativechanges in gene expression was performed by using the2minusΔΔCt method [36] with wheat glyceraldehyde-3-phosphatedehydrogenase (GAPDH) used as the endogenous control

Correlation between results from the array data and qRT-PCR was tested using Pearson correlation coefficient R [37]

3 Results and Discussion

31 Microarray Analysis As the most marked biologicaleffect of As

2O345x was observed in shoot length [30 31]

transcriptome analyses were performed on the coleoptiles of7-day-old wheat plantlets

Among a total of 61127 probe sets contained in themicroarrays 40398 (66) produced detectable hybridiza-tion signals under our experimental conditions

To identify genes expressed differentially the twofold cut-off criterion (log2 fold change gt1 and lt minus1) was adopted[38] Annotations were assigned to all of the genes thatexhibited differential transcript accumulation according tofour different groups of comparisons (Table 3) three compar-isons (treated control versus control poisoned-treated versuscontrol and poisoned-treated versus poisoned) were doneto identify genes differentially regulated in response to theapplication of ultrahigh diluted As

2O3in both unpoisoned

and poisoned systems A further comparison (poisonedversus control) was investigated to highlight genes involvedin seedling growth as a response of arsenic poisoning of seeds

According to the adopted criteria a total of 592 747 378and 643 probe sets were identified as differentially regulatedin the comparisons between C + T and C + 0 P + 0 and C + 0P + T and C + 0 and P + T and P + 0 respectively (Table 3)

When comparing the growth in water of poisoned (P + 0)and unpoisoned (C + 0) seeds it is evident that most of thegenes (85) were upregulated (635 out of 747) By growingpoisoned seeds in the presence of As

2O345x (P + T) instead

of distilled water the number of involved probe sets wasstrongly reduced (378) with 332 up- and 46 downregulatedprobe sets It seemed that the treatment of poisoned seedswith ultrahigh dilutedAs

2O3induced a normalization of gene

expression by bringing it closer to the basal levels usuallyoccurring in the control plantlets (C + 0)

In the unpoisoned system (C + T versus C + 0) a totalof 592 probe sets were differentially expressed and amongthem 71 (421 probes) were downregulated indicating thatthe growth of seedlings in the presence of As

2O345x

induced a general underexpression of the majority of affectedgenes A similar trend was observed in the poisoned system(P + T versus P + 0) where 65 of transcripts showed


Table 2 Gene ID and respective forward and reverse primernucleotide sequences (51015840ndash31015840) used for amplification by qRT-PCRFunctional categories of the genes are detailed in Table 1

Gene ID Primer sequence 51015840 rarr 31015840

Wheat GAPDH TTGCTCTGAACGACCATTTCGACACCATCCACATTTATTCTTC

BG909536 TGCCTGGCGCTTGCACCAGTACGAGGACCAAATCGA

BJ270709 TGCGGCTGGTTTCAAGTCACCCGGACGAGAGAAAACAAC

CK198840 GGCCGGCTTACGCATGTGGCCGTGGAGCTTACCATAA

CK207205 CATGGTCGGTTGCCATCAGTTCCCCAGTCAAGCTTTGGT

CK208220 GTAGCTGTCGAAGAACACATGCTTGCTCTGCGACACGAGGTACA

BQ838979 GCCCGTACGTGCAGTACAAACGTAGGCCCGCATCTTGTAG

CA635455 CCCGGCCTGGGAAGAATGGCTATGGCTTGGTTAGCA

CA660860 CCTGCTCGTCTCCCAGGATGCGCAGGCATCGTTTCTT

BE416616 TGCTCAAGATCTTCCTCGAGAACGGCGTGCTCGGTGTAGGT

BJ251672 GCAAGACGCTTCCTCTTCGTGTTGAAGCTTGTTCTTTTGCTTTG

BQ607338 CTCCTCCACCTCGATCCAAACATGGCTGACGGCTTCCTT

CA628396 AAGGGCTTGTTCGGCTAATTCCAAACCAATCTTTGCCAATCC

CD868502 TCCTCTTAGCCTGATGTGTTCATCCGACCACGACACGAACAAAC

CD869995 TCGCGACACCGCTTAGGTCAGAATCCAACCAACCAAAGCT

CA719316 CATCTTTAGCCATCCTCACAGAAATCTGTCAGATTCGTCGAGCAA

BJ225202 GGCCGTCACCAAGTTCACACCGCCATCAATCCATGAAA

BJ262694 TTGAAGTCCTGCGCGATCTAAGAGCACGGAGCTGCTGAT

BJ274465 TCACCCATGCTCGACGAATGAGATCCTGTATGTAATGCCTGCTA

BJ306445 GGATCCCATCTTAGTCCATGAGAGAGTAGCCGTCGTCCCCATA

BJ308450 GCGCAGGACACAGAGTGAAACGGTGTACTGATGGTTCCAGTTT

BJ320258 CAAGCTTGAGCACTCTCTGGAATCGTGTGCCTAGTGTCTGTGTTAA

BQ294672 CATAGCCAGCGCACTAGATTCAGGCTGACCCTGCTGTTTCC

BQ168973 CCAGGGCAACACGAGCTTTGCCAAACCCAATCCTAAGG

BJ322693 GCCACCAGGAGAGCAAACAGCGCTGCGGGTGATGAACT

CA613417 TGCCCGTTCTGCAGCAACGTCCCGTGTCCACTGTCT

Table 2 Continued


CA699507 CGGCACCACCTACTCATGTGCCATGGTCATTGGCGATAATC

CA708152 CCAGAAGCAGCGCAACGTGCGGTGCTTTTAGCCTTTAGG

CA708660 AAGCACCAAAAGAAGGCACAATCTTGGTGTCATGGGAGAAATTT

CK205521 GCCATCCCCGGTTCTTGGCTGCCGATCGCTTATGTC

BF484849 ACAGGTAACTGGGAGGGCAATAGATCTGAATTATGTGATGGGCATT

BJ226504 CTTCACCCTGCCCAAGGACCGCTGTTGCTGATCACAAA

BJ246114 GGCAGGCACACGAACGAGCGAGGGCTCTAGGTTACAACA

BJ303391 GCTGAAGAACCCGGAATCGTCCTCCTGGAATGCTGCAA

BJ312841 CGCATGACAGGAGAGCGTTTTGTCCCACTGACAGCAACAGTT

BQ578355 CCATCGCCTTCTACATCAAGGTGCGACGAGCTTCTCGATCTC

CA629836 TTGCACACCCTTCGACAAAGTTTGCTGCTTGCTCTTGAAAAA

CA640553 AACCCATGCGTCACCTGATCCCTACCTTCCATGAGCTCAACTG

CA681335 CTGGGAGTGCTGGTTGCATTGGCATGATAGGCTTCCTTGA

CA744534 CACGGATCTGGGACCTGAACCAGCGTGCCCTTCTAGCT

CA747224 CAGAGCAAACCCAACTCGAATACTTCTAGCCGCCATTTGGAA

CD453390 TCCGCGCTTGTTCAAGTACTCTTGGGTGCTGCCTTCCA

CK203324 TCTGAGCCCGGCACCATTGACGTTCCTGCCGATGTC

CK210556 AGAAGATGCGTGCGGTTCTCGTTCGGCCTCACCATTGG

BJ228692 GGCCTTCTTGGCCATCCTTACTTGCCCGGGAGATAAATCA

BJ243882 CAGGAAGCAGATCGCTGTTGAGGATGGCTACGTTCTGGTTTC

BJ256146 GGATAGGGCAGCTGCTTTCAGGCTTGACAGCCACCTTAGG

CA599873 CATGAGGACACCATGAGCAAAACCAGCATCCAGAATACCAGAAG

CA700404 ACTACCGCCTCCCCAACACCCGCGACTCGAAGAAGAAGT

CA731980 AGCGGCAGCTACGTGAACTTTGGGCTCCTCCACCATGA

CA642191 GGTAGAGAGGATCGCTGAAATCAGCCGCCAGCTTAGGAACTT

CA676257 TGGCAAACGCCGAGTACAGCGCCCAGTTGAAGCAGTA

CA676553 TTACCCCGCCAAAAAATGTATTACGGCTGCCTGCACATCT


Table 2 Continued


CA685752 GCTGGAGAGACCGTTTTGTCTTCGGTTTCGGCCTGTGATTTA

CA719198 CACCGCCAGAGGGTCTTTCCGCACTGCTCGATGATATGC

CK198851 GCCGGGCCCTTACGAATCCTTCAAGGTGGCAACAGGTT

Table 3 Number of upregulated downregulated and total regu-lated probe sets of the four investigated comparisons

Comparison Upregulated Downregulated TotalC + T versus C + 0 171 421 592P + 0 versus C + 0 633 114 747P + T versus C + 0 332 46 378P + T versus P + 0 224 419 643C + 0 = control C + T = treated control P + 0 = poisoned P + T = poisoned-treated

a downregulation in response to As2O345x application as

compared to waterComparison of the affected transcripts between specific

interactions was considered in order to differentiate commonandor group specific probe sets

In the poisoned system the comparison between P + 0versus C + 0 and P +T versus C + 0 groups (Figure 1) revealed146 probe sets common to both interactions whereas 601 and232 transcripts were specifically regulated in seedlings grownin water and As

2O345x respectively This suggested that the

application of ultrahigh diluted arsenic besides inducing afewer number of genes influenced the expression of sets ofgenes different from those involved in the growth of seedlingsin the presence of water Analogously by considering thecomparison between poisoned and unpoisoned seeds grownwith ultrahigh diluted arsenic in relation to their respectivecontrols grown in water (C + T versus C + 0 and P + T versusP + 0) 169 probe sets were common for both interactionswhereas 423 and 474 genes were specifically induced in theC + T versus C + 0 and P + T versus P + 0 interactionsrespectively (Figure 2)

The observed gene expression variation induced byUHDs is in line with other literature reports The regula-tion of gene expression as mechanism of action of UHDshas long been advocated by Khuda-Bukhsh and coworkers[14 16 39 40] on the basis of various scientific evidencesfrom both the higher organisms like mammals and lowerorganisms like yeasts and bacteria According to this gene-regulatory hypothesis ultrahigh diluted remedies carry spe-cific ldquosignalsrdquoldquoinformationrdquo that can be identified by specificreceptors of the cells These ldquosignalsrdquo can act as a ldquotriggerrdquofor turning ldquoonrdquo or ldquooffrdquo some relevant genes initiatinga cascade of gene actions to alter and correct the geneexpressions that went wrong to produce the disorderdiseaseAnother recent speculation brings about the hypothesis ofan epigenetic action (that affects gene expression rather thangene structure) of UHDs via water-mediated electromagnetic

P + 0

P + T versus C + 0

versus C + 0

601 146 232

Figure 1 Venn diagrams showing the number of gene transcriptsin poisoned (P + 0) and poisoned-treated (P + T) seedlings ascompared to the control (C + 0)

423 169 474

C + T versus C + 0 P + T versus P + 0

Figure 2 Venn diagrams showing the number of gene transcriptsin seedlings grown in ultrahigh diluted As

2O3as compared to their

respective control in both poisoned (P + T versus P + 0) andunpoisoned (C + T versus C + 0) experimental sets

information transfer of specific molecular signals [41] Theability of potentized homeopathic drugs to causemodulatehistone acetylationdeacetylation activities in some cancercells has recently been observed [19] These results stronglyvindicate the gene regulatory hypothesis that the potentizedhomeopathic drugs actually work through regulation ofexpression of relevant genes particularly through epigeneticmodifications [19]

32 Pathways To have a wider picture of the molecularmechanisms occurring at cellular level the microarray datawere investigated for effects on expression of genes belongingto known biochemical pathways or cellular processes in orderto gain better insight into the impact of ultrahigh dilutedAs2O3on wheat seedling growth

To this aim a custom MapMan image pathway wasgenerated and used for visualizing probe sets induced andorrepressed in four pairwise comparisons treated control (C +T) versus control (C + 0) (Figure 3) poisoned (P + 0) versuscontrol (C + 0) (Figure 4) poisoned-treated (P + T) versuspoisoned (P + 0) (Figure 5) and poisoned-treated (P + T)versus control (C + 0) (Figure 6)


DNA

SynthesisSynthesis

Regulation

Processing

Secondary metabolism

Hormone metabolism

Primary metabolism CHO

Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

PhotosynthesisUnknown

Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic

Biodegradation Metal handling

Mitochondrial e-transport

Repair

DNA

SynthesisSynthesis

Regulation

Processing


Hormonemetabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynthesis

Wall

Cell

Organization

Transport

Developmepp nt

Regulation

Stress

Biotic

AbioticAbiotic

Biodegradation Metalhandling

Unknown


Repair

minus3

minus2

minus1

0

3

2

1

Figure 3 Effect of ultrahigh diluted arsenic on wheat coleoptile gene expression (C + T versus C + 0) Gene expression changes aregraphically depicted by MapMan format version 351 in the frame of a custom-designed cell metabolism overview Each single squaregraphically represents one gene identified by microarray analysis and involved in the specific process illustrated The log2 color scalerepresents the mean expression ratio (poisonedcontrol) and ranges from minus3 (dark blue) downregulated to +3 (dark red) upregulatedThe complete dataset used for MapMan analysis and related details are given in Table S3 (see Supplementary Material available online athttpdxdoiorg1011552014851263)

Probe sets differentially expressed in the studied groupsof comparisonwere sorted into different functional categoriesrepresentative of the main cellular processes genes involvedin primary (aminoacid lipid nucleotide fermentationcofactors and vitamins tricarboxylic acid transformationand carbohydrates) secondary and hormone metabolismphotosynthesis cell wall mitochondrial e-transport metalhandling and xenobiotic biodegradation genes that encodestress related proteins (biotic and abiotic) genes involved ingeneral cell activities (organization transport developmentand signaling) genes encoding for enzymes genes involvedin protein metabolism (synthesis modification degradationand targeting) and genes associated with RNA (synthesisregulation and processing) and DNA (synthesis repair)regulation mechanisms

Most classes of those genes are known to be inducedduring plant response to environmental stresses and encodefor products (i) that directly protect plant cells againststresses such as heat stress proteins (HSPs) or chaperones lateembryogenesis abundant (LEA) proteins osmoprotectantsdetoxification enzymes and free-radical scavengers such assecondary metabolites (ii) that are involved in signaling

cascades and in transcriptional control such as kinaseshistone proteins and transcriptional factors and (iii) that areinvolved in general metabolic functions (lipid and proteinmetabolism)

321 Poisoned (P + 0) and Poisoned-Treated (P + T) versusControl (C + 0) Seedlings In our study the effect of seedpoisoning with arsenic trioxide was inferred by consideringthe transcriptome response of poisoned (P + 0) versus control(C + 0) experimental sets For this comparison a total of743 data points were represented in the custom MapManimage pathway (Figure 4) The majority of probe sets wereconsistently upregulated highlighting the activation of amyriad of biochemical responses aimed at contrasting theeffects of seed arsenic exposure By considering insteadpoisoned-treated (P + T) versus control (C + 0) plants theeffect of ultrahigh diluted arsenic on seedling transcriptomeprofile could be hypothesized

In both groups of comparison our microarray datasuggested the regulation of various genes involved in cellprimary metabolism (Tables S1 and S2) In the poisonedgroup (P + 0) almost all probe sets were upregulated


DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1

Figure 4 Effect of wheat seed poisoning with a sublethal dose of As2O3(5mM) on coleoptile gene expression (P + 0 versus C + 0) Gene

expression changes are as Figure 3 The complete dataset used for MapMan analysis and related details are given in Table S1

indicating a concerted cell response to arsenic poisoningSeveral evidences demonstrate that plant exposure to arsenicdoes result in the generation of reactive oxygen species(ROS) that can directly damage proteins amino acids andnucleic acids and cause membrane lipid peroxidation [42ndash47] In our study most of the genes were associated withaminoacid and lipid metabolism In particular 13 probe setswere involved in aminoacid synthesis pathways and wereoverexpressed two other genes were related to tryptophandegradation with one of them being overexpressed and theother one downregulated The same diffuse upregulationwas observed for genes involved in lipid metabolism Theseincluded 19 probe sets encoding for enzymes undergoing thesynthesis of fatty acids and phospholipids as well as lipiddegradation enzymes such as lipases Other genes involved innucleotide synthesis carbohydrate (CHO) metabolism tri-carboxylic acid (TCA) transformation cofactor and vitaminmetabolism and fermentation pathways were also positivelyinduced in the P + 0 group

Conversely in the P + T group only the expressionof few genes resulted to be affected and particularly thoseinvolved in aminoacid metabolism fatty acid and phospho-lipid and nucleotide synthesis Fatty acids and phospholipidsare crucial components of cellular membranes suberin andcutin waxes that provide structural barriers to the environ-ment They contribute to inducible stress resistance through

the remodeling ofmembrane fluidity and the release throughlipase activity of 120572-linolenic acid and as modulators of plantdefense gene expression [48] Modification of membranefluidity is thus an important functional response of plantsduring general abiotic stress

Genes belonging to the subclasses fermentation TCA-transformation CHO cofactors and vitamins did not appearin the map of the P + T group indicating that their expressionwas comparable with that of the control group

A differential regulation of genes encoding for stress-related proteins involved in the response to biotic and abioticstimuli was also observed Most of the genes encodingfor various heat-shock cold and droughtsalt responsiveproteins were consistently upregulated in the poisoned groupas compared to the control Those same genes were notaffected in the poisoned-treated group where the treatmentwith As

2O345x seemed to bring the levels of gene expression

close to those of the control The synthesis of such proteinshas been reported to increase after various forms of abioticstress [49] These molecular chaperones function by helpingin the folding of nascent polypeptide chains the refoldingof denatured proteins and the prevention of irreversibleprotein aggregation and insolubilization [50] They increasethe rate of folding and thus increase the resistance of cellsunder stress conditions


DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1

Figure 5 Effect of ultrahigh diluted arsenic on wheat coleoptile gene expression (P + T versus P + 0) grown from seeds poisoned with asublethal dose of As

2O3(5mM) Gene expression changes are as Figure 3The complete dataset used for MapMan analysis and related details

are given in Table S4

Other stress-related proteins are those involved in bioticagents recognition This recognition activates signal trans-duction cascades that lead to the activation of plant defensemechanisms In our study we observed the modulation ofgenes encoding disease-related proteins that were both up-and downregulated in the P + 0 In the P + T group theprobe sets encoding for proteins related to biotic stresswere mostly upregulated The activation of pathogen-relatedpathways could sound senseless as the stressor applied in ourwheat model is of abiotic origin however a growing body ofevidence supports the notion that plant signaling pathwaysconsist of elaborate networks with frequent crosstalk therebyallowing plants to regulate both abiotic stress tolerance anddisease resistance [51]

Among the negative effects on plants induced by arsenica reduction of the photosynthesis rate was observed by otherauthors [52] Arsenic damaged the chloroplast membraneand disorganized the membrane structure inducing func-tional changes of the integral photosynthetic process Thetreatment of seeds with As

2O3in our wheat model (P + 0)

turned into an upregulation of some of the genes encoding forproteins of the photosystems ATP synthase and enzymes ofthe Calvin cycle Beside one probe set encoding for photosys-tem polypeptides all those genes resulted to be not inducedin the P + T group as compared to the controlThis suggested

that in seedlings poisoned and then grown in the presenceof ultrahigh diluted arsenic the photosynthetic processesoccurred similar to those of the control plants (C + 0)

The effects of arsenic-poisoning were also evident ongenes involved in nucleic acid synthesis regulation andprocessing Sevendifferent histones (the chromatin structuralproteins) together with two DNA repair factors were upregu-lated (log2 fold change gt11) in the P + 0 group Fewer histonegenes were involved in the response of the P + T groupand among them one was even downregulated as comparedto the control plants The same trend was observed forRNA where against a diffuse upregulation of genes involvedin transcription regulation of transcription and processingobserved (for a total of 24 probe sets) in P + 0 plants onlyfew genes involved mainly in RNA transcription regulationwere positively modulated in the P + T group

Histone modifications are among the epigenetic mech-anisms regulating gene expression in response to devel-opmental and environmental stimuli [53] Since epigeneticmodifications do not affect the DNA sequence these changesshow the potential of plasticity and underlie environmentalinfluences Recent works demonstrated that UHDs emit elec-tromagnetic signals [54] indicating the suggestive hypothesisof an epigenetic action of UHDs on biological systems [41]The ability of ultrahigh diluted remedies to trigger epigenetic


DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing

Secondary metabo

Hormmetab


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynth

Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

AbioticAbiotic


Unknown

Mitochondritransport

Repair

minus3

minus2

minus1

0

3

2

1

Figure 6 Effect of ultrahigh diluted arsenic onwheat coleoptile gene expression (P + T versus C + 0) grown from poisoned seeds as comparedto unpoisoned seeds The complete dataset used for MapMan analysis and related details are given in Table S2

modifications like methylationdemethylation of DNA andacetylationdeacetylation of histone has also been recentlydemonstrated in some cancer cells [55]

The involvement of genes encoding for metal bindingproteins and factors related to xenobiotic biodegradationwere also observed in poisoned seedlings after As

2O345x

treatment (P + T group) As those genes were slightly inducedor even negatively modulated in the poisoned group aspecific upregulation induced by ultrahigh diluted arsenicof metal-handling targeted defense mechanisms could behypothesized

As regards cell activities various genes encoding pro-teins involved in organization transport development andsignaling mechanisms exhibited altered expression levels inresponse to seed poisoningwithAs

2O3 Among cell transport

systems ATP-binding cassette (ABC) transporters mediatethe translocation of a wide range of structurally unrelatedmolecules across biological membranes [56 57] In ourstudy ABC transporters and multidrug resistance proteinswere specifically activated in the poisoned group Thosekinds of transporters were not present in the P + T groupindicating that they were expressed at the levels found inthe control plants (C + 0) As regards signaling moleculesprotein kinases which were upregulated in seedlings of thepoisoned group (P + 0) are known to play crucial rolesin signal transduction pathways in all eukaryotes Some

kinase genes were also overexpressed in seedlings grownwith As

2O345x suggesting the importance of those signal

proteins in response to environmental stimuli Literaturereports suggested that the oxidative tissue damage inducedby heavy metals [58 59] could result also in the elevation ofCa2+ concentration in plant cells in which Ca2+ played animportant role in free radical scavenger induction [60 61]

Three probe sets encoding for calcium-related transportand signaling systems were activated in response to arsenicpoisoning of seeds (P + 0) whereas in P + T those probe setswere not affected suggesting again a reequilibration of geneexpression induced by As

2O345x to the levels of the control

(C + 0)Among cell signaling molecules hormone-related pro-

teins are known to have a key role in the pathways thatgovern biotic and abiotic stress responses [51] Our resultsshowed the involvement of some genes encoding for proteinsresponsive to auxins gibberellins ethylene jasmonate andabscisic acid that resulted to be moderately overexpressed(log2 FC from 10 to 13) in the poisoned group (P + 0)The same trend was observed in the P + T group wherea more marked upregulation (log2 FC from 13 to 28) ofthose hormone-related proteins was observed According tothose observations a strengthening effect in the hormone-mediated defense response of poisoned seedlings may beattributed to the As

2O3treatment Other hormone-related


genes (mainly responsive to brassinosteroids) were signifi-cantly downregulated (log2 FC from minus12 to minus23) in bothP + 0 and P + T groups Brassinosteroids have a critical rolein plant growth regulation including stem elongation [62]Their negative modulation in poisoned and poisoned-treatedseedlings as compared to control is consistent with shortercoleoptile lengths recorded in the biological observations

Another important functional class generally involvedin plant response to biotic and abiotic stresses is sec-ondary metabolites Genes involved in the metabolism ofisoprenoids phenylpropanoids phenols and wax were con-sistently induced (log2 FC from 10 to 26) in the poisonedgroupThis is in line with other findings reporting the stress-protective role of thosemetabolites inmitigating arsenic toxiceffects [63] Conversely only three probe sets involved inflavonoid metabolism were differentially expressed in theP + T group and all of them resulted to be downregulated ascompared to the control (C + 0)

The observed downregulation along with the fact thatgenes induced in the poisoned group were not present inthe poisoned-treated group may suggest a putative effect ofultrahigh diluted As

2O3in bringing the metabolic situation

of poisoned seedlings close to that of the control plantsProteolysis plays an important role in stress response

pathways One important function is the removal of damagedproteins to avoid accumulation as potentially harmful aggre-gates and to eliminate proteins with compromised activity[64]

On the whole we observed the upregulation of manygenes encoding for proteases peptidases proteasome andubiquitin systems in the response of P + 0 plants following theapplication of an oxidative stress (sublethal dose of arsenic)This upregulation was not confirmed for most of the genes inthe P + T group where gene expression was comparable oreven downregulated as compared to the control group

Other genes involved in protein metabolism such assynthesis posttranslational modification and targeting werefound to be markedly upregulated in the poisoned group Asobserved for protein degradation only a limited number ofgenes belonging to the above listed functional subclasses wereactivated in the poisoned-treated group whereas all otherprobe sets had expression levels close to those observed inthe control

Gene expression analysis revealed that among cell detox-ification systems most cytochrome P450 enzymes peroxi-dases and glutathione transport proteins were strictly regu-lated in the response of the plant to several abiotic stressorssuch as UV damage heavy metal toxicity mechanical injurydrought high salinity and low temperatures [65]

In our study most of the genes encoding for cytochromeP450 proteins were strongly upregulated (from 11 to 34 log2fold changes) in the P + 0 group whereas those same geneswere not induced in poisoned seeds grown with As

2O345x

(P + T) indicating that expression values were comparablewith those of the control group (C + 0)

Intuitively response to conditions associated with oxida-tive stress must in part rely on endogenous antioxidativedefense mechanisms required tomaintain cellular homeosta-sis Glutathione-S transferases (GSTs) are ubiquitous proteins

in plants that play important roles in stress tolerance anddetoxification metabolism Detoxification of xenobiotics isconsidered to be the main function of plant GSTs but otherfunctions include protecting cells from a wide range ofbiotic and abiotic stressors including pathogen attack heavymetal toxins oxidative stress and UV radiation [66ndash68]However in our study only tree probes encoding for GSTswere involved one in P + 0 group (underexpressed) and twoin the P + T group (one downregulated and one upregulated)This observation clearly contradicts our expectation about adiffuse GST induction in response to the imposed oxidativestress Considering that our experimental end-point was at7-day after treatment it is probable that changes in GSTtranscript abundance did occur only within the first hoursafter stress application as observed by other authors [69 70]

Peroxidases are a group of enzymes located in vacuolecell wall thylakoid membranes and extracellular space thatare involved in plant defense against reactive oxygen species

Our microarray data revealed a significant underexpres-sion for one ormore probe sets in both P + 0 and P +T groupsas compared to the control This downregulation of peroxi-dases was also observed by Chakrabarty et al [71] reportingthe effects of arsenate and arsenite stresses on rice seedlingsAccording to their observations peroxidases downregulationwas correlated with the activation of a chloroplast-targetedlipoxygenase that presumably caused a damage to thylakoidmembranes In our study along with the downregulation ofperoxidases the activation of cell wall degradation systemswas also observed suggesting an arsenic-induced damage ofcell wall components

322 Treated Control (C + T) versus Control (C + 0) andPoisoned-Treated (P + T) versus Poisoned (P + 0) Seedlings Ifwe consider the two interactions ldquotreated control (C + T) vscontrol (C + 0)rdquo and ldquopoisoned treated (P + T) vs poisoned(P + 0)rdquo a clear effect of ultra-high diluted As

2O3in two

distinct systems (poisoned and unpoisoned) can be observedFrom Figures 3 and 6 the occurrence of an overall reductionof gene expression of most of the involved probe sets can bededuced Literature evidences demonstrated that elicitors caninduce a downregulation of the transcript levels of severalgenes concomitant with the induction of a defense responsein plants Therefore the downregulation of gene expressioncan be also associated with defense responses [72]

In the context of this massive gene underexpressionsome gene categories resulted to be upregulated in bothsystems (unpoisoned and poisoned) (Tables S3 and S4) Itis the case of genes grouped under the functional categoryldquohormone metabolismrdquo in the C + T versus C + 0 groupthe upregulation involved genes responsive to auxins brassi-nosteroids and jasmonate whereas in the P + T versus P +0 group a consistent overexpression of abscisic acid andethylene-regulated genes was observed This suggested thatin some way ultrahigh diluted As

2O3activated hormone-

mediated response pathways independently of whether thesystems had been poisoned or not A similar behavior wasobserved for some genes involved in the phenylpropanoidpathway particularly for probe sets encoding for the enzyme


phenylalanine ammonia lyase (PAL)The activation of stress-responsive hormones and secondary metabolism has beenalready observed for several plant species where stress hor-mones like jasmonic acid or ethylene resulted to mediateelicitor-induced accumulation of secondary metabolites [73]It is presumable that ultrahigh diluted As

2O3acted like a sort

of elicitor that induced a plant response in terms of selectiveactivation of specific pathways (hormone-mediated and sec-ondary metabolites) along with a general downregulation ofmost other genes

Other upregulations were observed even if not sharedby both systems In the C + T versus C + 0 group variousprobe sets encoding for protein kinases were activatedalong with other genes involved in protein modificationand degradation Protein interaction andor modification isthe best way for the cell to strictly regulate the activity oftranscription factors in signal transduction pathways thatrequire rapid gene induction [74] It is thus highly probablethat ultrahigh diluted As

2O3represented a kind of signal for

the cell in the unpoisoned systemIn the P + T versus P + 0 group the upregulation

concerned genes involved in metal handling mitochon-drial electron transport and biotic stress whose inductionwas presumably part of the plant recovery mechanism inresponse to the stress imposed at the system (seed poisonedwith As

2O3) The observation that ultrahigh diluted As

2O3

induced a response in the unpoisoned seedlings (C +T versusC + 0) comparable with the trend observed in the poisonedsystem (P+T versus P+ 0) suggested that the artificial growthof wheat did impart a sort of stress to the system indicatingthat in vitro conditions are by nomeans the perfect substitutesfor the natural ones

The results of the present study clearly demonstratedthat the expression profiles of several classes of genes inplants treated with ultrahigh diluted As

2O3were significantly

different from that of the placebo (water) treated plants Thepresent microarray data in experimental wheat genome lendfurther critical support to the gene regulatory hypothesisfirst proposed by Khuda-Bukhsh [14 16] Several reportsby Khuda-Bukhsh and coworkers revealed quite clearly thatcertain ultrahigh diluted homeopathic remedies were capableof altering gene expression while the placebos invariablyfailed to do so this became more evident with the results onthe ability of potentized homeopathic drugs to trigger keyepigenetic events like methylationdemethylation of DNAand acetylationdeacetylation of histone [55] which are anintegral part of the epigenetic modification route [19] How-ever how a homeopathic remedy diluted beyond Avogadrorsquoslimit can elicit response in a cell and bind with the receptoris not yet precisely known Some recent lines of evidencesuggest that certain changes in physical-chemical parametersof the solvent occur when homeopathic drugs are potentizedby the procedure of successions and serial dilutions [19] andnanoparticles of the same drug are still retained in the diluentwith their medicinal property preserved [19 75] How thistranslates into changes in biological systems is presently at aspeculative stage and needs to be further explored [19]

The possible pathways and sites of action of UHDshave also been discussed by Khuda-Bukhsh and coworkers

[24 76ndash79] Presumably their action is mediated throughcytokine responses Thus administration of UHDs can elicitresponse in suitable signal proteins and can either upregulateor downregulate such signal proteins to bring back therecovery of the patient to normal health [42]

33 Gene Expression Analysis Using qRT-PCR Real-timePCR is generally considered the ldquogold-standardrdquo assay formeasuring gene expression and is often used to confirmfindings frommicroarray data A total of 55 probe sets encod-ing for genes involved in stress-related functional categories(lipid metabolism histones and cellular structural proteinsstress proteins cell signaling transduction and transportproteins secondary metabolism protein metabolism andcell detoxification systems) and that resulted to be over-or underexpressed in the four investigated groups wererandomly selected for real-time PCR validation

The gene expression profiles determined by microarrayand real-time PCR were found to be well correlated 119875 lt 005(1198772 = 077)This indicated thatmicroarray data were reliableThe correlation coefficient of 077 is very good consideringthat microarray data are semiquantitative and subject toerror for multigene families where different transcripts couldhybridize to similar probes on the array qRT-PCR data aremore specific since the amplification of single transcripts isconfirmed by melting curves

4 Conclusions

In this study the analysis of the transcriptome profile ofwheatcoleoptiles grown from arsenic poisoned seeds suggested theinvolvement of a complex network of regulatory pathways inAs-response Along with genes harboring general metabolicfunctions the changes in expression profiles of many knowngenes involved in or affected by general abiotic stresses wereobserved

The effect of ultrahigh diluted As2O3was particularly

striking on the poisoned system a reduction of gene expres-sion levels to values comparable to those of the control(water-treated) was observed for several functional classes ofgenes It was the case of probe sets involved in cytochromeP450 histones stress-related protein synthesis and otherimportant functional classes implicated in cell response toenvironmental stimuli This suggested complex and con-certed actions of the cell involving most metabolic functionsfrom genome rearrangement to proteome and metabolomemodification resulting in final cell survival

The understanding of how these genes are connectedwitheach other and how they are linked to biological responsesmust be further investigated A plausible hypothesis is thatAs2O345x treatment induced a reequilibration of those genes

that were upregulated in the poisoned group by bringingtheir gene expression levels closer to the basal levels usuallyoccurring in the control plants We cannot surely affirmwhether this response is advantageous or not for the plantbut the biological measurements of plant shoots and roots[30 31] seemed to confirm a vigorous growth of As 45x-treated seedlings as compared to water-treated seedlings


The present work should be considered as the first stepof a science-based route towards the comprehension of UHDremedial effects So far UHD possible mechanisms of actionremain intangible theories and it will be important ultimatelyto substantiate these with compelling research evidence

The findings of the present study contributed to supportthe gene regulatory hypothesis first proposed by Khuda-Bukhsh [16] However the actual molecular mechanisminvolved in transmission of information of the UHD down tothe execution level has to be more clearly ascertained [19] Inour opinion further investigations integrating the genomicproteomic and metabolomic approaches could shed lightonto the molecular responses triggered by UHDs not only inplants but also in humans

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by Laboratoires BoironLyon France The authors wish to sincerely thank DrStephane Caredda for his encouragement and support

References

[1] B Marschollek M Nelle M Wolf S Baumgartner P Heusserand U Wolf ldquoEffects of exposure to physical factors onhomeopathic preparations as determined by ultraviolet lightspectroscopyrdquo TheScientificWorldJournal vol 10 pp 49ndash612010

[2] P Bellavite and L Betti ldquoHomeopathy and the science ofhigh dilutions when to believe the unbelievablerdquo InternationalJournal of High Dilution Research vol 11 no 40 pp 107ndash1092012

[3] M Brizzi D Nani M Peruzzi and L Betti ldquoStatistical analysisof the effect of high dilutions of arsenic in a large dataset froma wheat germination modelrdquo British Homeopathic Journal vol89 no 2 pp 63ndash67 2000

[4] M Brizzi V Elia G Trebbi D Nani M Peruzzi and LBetti ldquoThe efficacy of ultramolecular aqueous dilutions on awheat germinationmodel as a function of heat and aging-timerdquoEvidence-Based Complementary and Alternative Medicine vol2011 Article ID 696298 11 pages 2011

[5] C M Witt M Bluth H Albrecht T E R Weiszlighuhn SBaumgartner and S N Willich ldquoThe in vitro evidence for aneffect of high homeopathic potencies-A systematic review of theliteraturerdquo Complementary Therapies in Medicine vol 15 no 2pp 128ndash138 2007

[6] P C Endler W Matzer C Reich et al ldquoSeasonal variation ofthe effect of extremely diluted agitated gibberellic acid (10119890minus30)on wheat stalk growth a multiresearcher studyrdquo The ScientificWorld Journal vol 11 pp 1667ndash1678 2011

[7] P Bellavite R Ortolani and A Conforti ldquoImmunology andhomeopathy 3 Experimental studies on animal modelsrdquoEvidence-based Complementary and Alternative Medicine vol3 no 2 pp 171ndash186 2006

[8] S BaumgartnerD Shah J Schaller UKampfer AThurneysenand P Heusser ldquoReproducibility of dwarf pea shoot growthstimulation by homeopathic potencies of gibberellic acidrdquoComplementaryTherapies inMedicine vol 16 no 4 pp 183ndash1912008

[9] D Shah-Rossi P Heusser and S Baumgartner ldquoHomeopathictreatment of Arabidopsis thaliana plants infected with Pseu-domonas syringaerdquo TheScientificWorldJournal vol 9 pp 320ndash330 2009

[10] L Betti G Trebbi M Zurla D Nani M Peruzzi andM BrizzildquoA review of three simple plant models and correspondingstatistical tools for basic research in homeopathyrdquoThe ScientificWorld Journal vol 10 pp 2330ndash2347 2010

[11] T Jager C Scherr M Simon P Heusser and S Baumgart-ner ldquoEffects of homeopathic arsenicum album nosode andgibberellic acid preparations on the growth rate of arsenic-impaired duckweed (Lemna gibba L)rdquo The Scientific WorldJournal vol 10 pp 2112ndash2129 2010

[12] P Magnani A Conforti E Zanolin M Marzotto and PBellavite ldquoDose-effect study ofGelsemium sempervirens in highdilutions on anxiety-related responses in micerdquo Psychopharma-cology vol 210 no 4 pp 533ndash545 2010

[13] B Stock-Schroer H Albrecht L Betti et al ldquoReporting exper-iments in homeopathic basic research (REHBaR)mdasha detailedguideline for authorsrdquo Homeopathy vol 98 no 4 pp 287ndash2982009

[14] A R Khuda-Bukhsh ldquoTowards understanding molecularmechanisms of action of homeopathic drugs an overviewrdquoMolecular and Cellular Biochemistry vol 253 no 1-2 pp 339ndash345 2003

[15] S Baumgartner P Heusser and A Thurneysen ldquoMethod-ological standards and problems in preclinical homoeopathicpotency researchrdquo Forschende Komplementarmedizin vol 5 no1 pp 27ndash32 1998

[16] A Khuda-Bukhsh ldquoPotentized homoeopathic drugs actthrough regulation of gene-expression a hypothesis toexplain their mechanism and pathways of action in vitrordquoComplementary Therapies in Medicine vol 5 no 1 pp 43ndash461997

[17] A De D Das S Dutta D Chakraborty N Boujedaini and AR Khuda-Bukhsh ldquoPotentized homeopathic drug Arsenicumalbum 30C inhibits intracellular reactive oxygen species gener-ation and up-regulates expression of arsenic resistance gene inarsenine-exposed bacteria Escherichia colirdquo Journal of ChineseIntegrative Medicine vol 10 no 2 pp 210ndash227 2012

[18] D Das A De S Dutta R Biswas N Boujedaini and AR Khuda-Bukhsh ldquoPotentized homeopathic drug ArsenicumAlbum 30C positively modulates protein biomarkers and geneexpressions in Saccharomyces cerevisae exposed to arsenaterdquoJournal of Chinese IntegrativeMedicine vol 9 no 7 pp 752ndash7602011

[19] A R Khuda-Bukhsh ldquoCurrent trends in high dilution researchwith particular reference to gene regulatory hypothesisrdquo TheNucleus vol 57 no 1 pp 3ndash17 2014

[20] P Chavan K Joshi and B Patwardhan ldquoDNA microarraysin herbal drug researchrdquo Evidence-based Complementary andAlternative Medicine vol 3 no 4 pp 447ndash457 2006

[21] E Bigagli C Luceri S Bernardini A Dei and P DolaraldquoExtremely low copper concentrations affect gene expressionprofiles of human prostate epithelial cell linesrdquo Chemico-Biological Interactions vol 188 no 1 pp 214ndash219 2010


[22] P Bellavite M Marzotto O Debora et al ldquoCellular and tran-scriptional responses of neurocytes following in vitro exposureto Gelsemium sempervirensrdquo International Journal of HighDilution Research vol 11 no 40 pp 113ndash114 2012

[23] C C De Oliveira S M De Oliveira V M Goes C M ProbstM A Krieger and D D F Buchi ldquoGene expression profiling ofmacrophages following mice treatment with an immunomodu-lator medicationrdquo Journal of Cellular Biochemistry vol 104 no4 pp 1364ndash1377 2008

[24] S K Saha S Roy and A R Khuda-Bukhsh ldquoEvidence insupport of gene regulatory hypothesis gene expression profilingmanifests homeopathy effect as more than placebordquo Interna-tional Journal of High Dilution Research vol 12 no 45 pp 162ndash167 2013

[25] L Betti F Borghini and D Nani ldquoPlant models for fundamen-tal research in homeopathyrdquoHomeopathy vol 92 no 3 pp 129ndash130 2003

[26] L Betti G Trebbi V Majewsky et al ldquoUse of homeopathicpreparations in phytopathological models and in field trials acritical reviewrdquo Homeopathy vol 98 no 4 pp 244ndash266 2009

[27] T Jager C Scherr D Shah et al ldquoUse of homeopathicpreparations in experimental studies with abiotically stressedplantsrdquo Homeopathy vol 100 no 4 pp 275ndash287 2011

[28] L Kolisko Physiologischer und physikalischer Nachweis derWirksamkeit kleinster Entitaten Arbeitsgem Anthroposophis-cher Arzte Stuttgart Germany 1961

[29] W Pongratz and P C Endler ldquoReappraisal of a classicalbotanical experiment in ultra high dilution research Energeticcoupling in a wheat modelrdquo in Ultra High Dilution Physiologyand Physics P C Endler and J Schulte Eds pp 19ndash26 KluwerAcademic Dordrecht The Netherlands 1994

[30] L Betti M Brizzi D Nani and M Peruzzi ldquoEffect of highdilutions of Arsenicum album on wheat seedlings from seedpoisonedwith the same substancerdquoBritishHomeopathy Journalvol 86 no 2 pp 86ndash89 1997

[31] M Brizzi L Lazzarato D Nani F Borghini M Peruzzi andL Betti ldquoA biostatistical insight into the As

2O3high dilution

effects on the rate and variability of wheat seedling growthrdquoForschende Komplementarmedizin vol 12 no 5 pp 227ndash2832005

[32] G K Smyth ldquoLinear models and empirical Bayes methods forassessing differential expression in microarray experimentsrdquoStatistical Applications in Genetics and Molecular Biology vol3 no 1 article 3 2004

[33] Y Benjamini and Y Hochberg ldquoControlling the false discoveryrate a practical and powerful approach to multiple testingrdquoJournal of the Royal Statistical Society Series B Methodologicalvol 57 no 1 pp 289ndash300 1995

[34] O Thimm O Blasing Y Gibon et al ldquoMAPMAN a user-driven tool to display genomics data sets onto diagrams ofmetabolic pathways and other biological processesrdquo PlantJournal vol 37 no 6 pp 914ndash939 2004

[35] B Usadel A Nagel OThimmet al ldquoExtension of the visualiza-tion tool MapMan to allow statistical analysis of arrays displayof coresponding genes and comparisonwith known responsesrdquoPlant Physiology vol 138 no 3 pp 1195ndash1204 2005

[36] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862119879 methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[37] K Pearson ldquoMathematical contributions to the theory ofevolution III Regression heredity and panmixiardquoPhilosophicalTransitions of Royal Society vol 187 pp 253ndash318 1896

[38] M B Eisen P T Spellman P O Brown and D Botstein ldquoClus-ter analysis and display of genome-wide expression patternsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 95 no 25 pp 14863ndash14868 1998

[39] A R Khuda-Bukhsh ldquoLaboratory research in homeopathyrdquoIntegrative Cancer Therapies vol 5 no 4 pp 1ndash14 2006

[40] A R Khuda-Bukhsh S S Bhattacharyya S Paul S DuttaN Boujedaini and P Belon ldquoModulation of signal proteins aplausible mechanism to explain how a potentized drug secalecor 30c diluted beyond Avogadrorsquos limit combats skin papil-loma in micerdquo Evidence-based Complementary and AlternativeMedicine vol 2011 Article ID 286320 12 pages 2011

[41] F Borghini G Dinelli I Marotti G Trebbi G Borghini and LBetti ldquoElectromagnetic Information Transfer (EMIT) by Ultrahigh Diluted (UHD) solutions the suggestive hypothesis ofan epigenetic actionrdquo International Journal of High DilutionResearch vol 11 pp 113ndash114 2012

[42] S Dho W Camusso M Mucciarelli and A Fusconi ldquoArsenatetoxicity on the apices of Pisum sativum L seedling roots effectson mitotic activity chromatin integrity and microtubulesrdquoEnvironmental and Experimental Botany vol 69 no 1 pp 17ndash23 2010

[43] M Shri S Kumar D Chakrabarty et al ldquoEffect of arsenicon growth oxidative stress and antioxidant system in riceseedlingsrdquo Ecotoxicology and Environmental Safety vol 72 no4 pp 1102ndash1110 2009

[44] A Lin X Zhang Y-G Zhu and F-J Zhao ldquoArsenate-inducedtoxicity effects on antioxidative enzymes and dna damage inVicia fabardquoEnvironmental Toxicology andChemistry vol 27 no2 pp 413ndash419 2008

[45] C-X Li S-L Feng Y Shao L-N Jiang X-Y Lu and X-LHou ldquoEffects of arsenic on seed germination and physiologicalactivities of wheat seedlingsrdquo Journal of Environmental Sciencesvol 19 no 6 pp 725ndash732 2007

[46] H P Singh D R Batish R K Kohli and K Arora ldquoArsenic-induced root growth inhibition inmung bean (Phaseolus aureusRoxb) is due to oxidative stress resulting from enhanced lipidperoxidationrdquo Plant Growth Regulation vol 53 no 1 pp 65ndash732007

[47] J Dat S Vandenabeele E Vranova M van Montagu D Inzeand F Van Breusegem ldquoDual action of the active oxygenspecies during plant stress responsesrdquo Cellular and MolecularLife Sciences vol 57 no 5 pp 779ndash795 2000

[48] R G Upchurch ldquoFatty acid unsaturation mobilization andregulation in the response of plants to stressrdquo BiotechnologyLetters vol 30 no 6 pp 967ndash977 2008

[49] W Wang B Vinocur O Shoseyov and A Altman ldquoRole ofplant heat-shock proteins and molecular chaperones in theabiotic stress responserdquo Trends in Plant Science vol 9 no 5 pp244ndash252 2004

[50] R Van Montfort C Slingsby and E Vierling ldquoStructure andfunction of the small heat shock protein120572-crystallin family ofmolecular chaperonesrdquo Advances in Protein Chemistry vol 59no 2 pp 105ndash156 2001

[51] M Fujita Y Fujita YNoutoshi et al ldquoCrosstalk between abioticand biotic stress responses a current view from the points ofconvergence in the stress signaling networksrdquo Current Opinionin Plant Biology vol 9 no 4 pp 436ndash442 2006

[52] E Miteva and M Merakchiyska ldquoResponse of chloroplasts andphotosynthetic mechanism of bean plants to excess arsenic insoilrdquo Bulgarian Journal of Agricultural Science vol 8 no 2-3pp 151ndash156 2002


[53] Y Zhu A Dong and W-H Shen ldquoHistone variants and chro-matin assembly in plant abiotic stress responsesrdquo Biochimica etBiophysica ActamdashGene Regulatory Mechanisms vol 1819 no 3-4 pp 343ndash348 2012

[54] L Montagnier J Aıssa S Ferris J-L Montagnier and CLavalleee ldquoElectromagnetic signals are produced by aqueousnanostructures derived from bacterial DNA sequencesrdquo Inter-disciplinary Sciences Computational Life Sciences vol 1 no 2pp 81ndash90 2009

[55] K Bishayee S Sikdar and A R Khuda-Bukhsh ldquoEvidence ofepigenetic modification in cell cycle arrest caused by the use ofultra-highly diluted Gonolobus condurango extractrdquo Journal ofPharmacopuncture vol 16 no 4 pp 7ndash13 2013

[56] C F Higgins ldquoABC Transporters from microorganisms tomanrdquo Annual Review of Cell Biology vol 8 pp 67ndash113 1992

[57] B I Holland S P C Cole K Kuchler and C F HigginsABC Proteins from Bacteria to Man Elsevier Amsterdam TheNetherlands 2002

[58] F Yu K Liu M Li Z Zhou H Deng and B Chen ldquoEffectsof cadmium on enzymatic and non-enzymatic antioxidativedefences of rice (Oryza sativa L)rdquo International Journal ofPhytoremediation vol 15 no 6 pp 513ndash521 2013

[59] S J Stohs and D Bagchi ldquoOxidative mechanisms in the toxicityof metal ionsrdquo Free Radical Biology and Medicine vol 18 no 2pp 321ndash336 1995

[60] P K Hepler ldquoCalcium a central regulator of plant growth anddevelopmentrdquo Plant Cell vol 17 no 8 pp 2142ndash2155 2005

[61] A H Price A Taylor S J Ripley A Griffiths A J Trewavasand M R Knight ldquoOxidative signals in tobacco increasecytosolic calciumrdquo The Plant Cell vol 12 no 9 pp 1387ndash13981994

[62] S D Clouse and J M Sasse ldquoBrassinosteroids essential regula-tors of plant growth and developmentrdquo Annual Review of PlantPhysiology and Plant Molecular Biology vol 49 pp 427ndash4511998

[63] P M Finnegan and W Chen ldquoArsenic toxicity the effects onplant metabolismrdquo Frontiers in Physiology vol 3 article 1822012

[64] K Flick and P Kaiser ldquoProtein degradation and the stressresponserdquo Seminars in Cell and Developmental Biology vol 23no 5 pp 515ndash522 2012

[65] Y Narusaka M Narusaka M Seki et al ldquoCrosstalk in theresponses to abiotic and biotic stresses in Arabidopsis analysisof gene expression in cytochrome P450 gene superfamily bycDNA microarrayrdquo Plant Molecular Biology vol 55 no 3 pp327ndash342 2004

[66] S C Kampranis R Damianova M Atallah et al ldquoA novel plantglutathione S-transferaseperoxidase suppresses Bax lethalityin yeastrdquo Journal of Biological Chemistry vol 275 no 38 pp29207ndash29216 2000

[67] L Loyall K Uchida S Braun M Furuya and H FrohnmeyerldquoGlutathione and a UV light-induced glutathione S-transferaseare involved in signaling to chalcone synthase in cell culturesrdquoThe Plant Cell vol 12 no 10 pp 1939ndash1950 2000

[68] L A Mueller C D Goodman R A Silady and V WalbotldquoAN9 a petunia glutathione S-transferase required for antho-cyanin sequestration is a flavonoid-binding proteinrdquo PlantPhysiology vol 123 no 4 pp 1561ndash1570 2000

[69] C Hoenemann S Richardt K Kruger A D Zimmer A Hoheand S A Rensing ldquoLarge impact of the apoplast on somaticembryogenesis in Cyclamen persicum offers possibilities for

improved developmental control in vitrordquo BMC Plant Biologyvol 10 article 77 2010

[70] H Zhu L Tu S Jin et al ldquoAnalysis of genes differentiallyexpressed during initial cellular dedifferentiation in cottonrdquoChinese Science Bulletin vol 53 no 23 pp 3666ndash3676 2008

[71] D Chakrabarty P K Trivedi P Misra et al ldquoComparativetranscriptome analysis of arsenate and arsenite stresses in riceseedlingsrdquo Chemosphere vol 74 no 5 pp 688ndash702 2009

[72] K Suzuki T Nishiuchi Y Nakayama M Ito and H ShinshildquoElicitor-induced down-regulation of cell cycle-related genes intobacco cellsrdquo Plant Cell and Environment vol 29 no 2 pp183ndash191 2006

[73] J Memelink R Verpoorte and J W Kijne ldquoORCAnization ofjasmonate-responsive gene expression in alkaloid metabolismrdquoTrends in Plant Science vol 6 no 5 pp 212ndash219 2001

[74] D Vom Endt J W Kijne and J Memelink ldquoTranscriptionfactors controlling plant secondary metabolism what regulatesthe regulatorsrdquo Phytochemistry vol 61 no 2 pp 107ndash114 2002

[75] P S Chikramane A K Suresh J R Bellare and S G KaneldquoExtreme homeopathic dilutions retain starting materials ananoparticulate perspectiverdquo Homeopathy vol 99 no 4 pp231ndash242 2010

[76] P Mallick J Chakrabarti Mallick B Guha and A R Khuda-Bukhsh ldquoAmeliorating effect of microdoses of a potentizedhomeopathic drug ArsenicumAlbum on arsenic-induced tox-icity in micerdquo BMC Complementary and Alternative Medicinevol 3 Article ID 7 13 pages 2003

[77] S Pathak J K Das S J Biswas and A R Khuda-BukhshldquoProtective potentials of a potentized homeopathic drugLycopodium-30 in ameliorating azo dye induced hepatocar-cinogenesis in micerdquo Molecular and Cellular Biochemistry vol285 no 1-2 pp 121ndash131 2006

[78] S Pathak N Bhattacharjee J K Das et al ldquoSupportiveevidence for the anticancerous potential of alternativemedicineagainst hepatocarcinogenesis in micerdquo Forschende Komple-mentarmedizin vol 14 no 3 pp 148ndash156 2007

[79] A R Khuda-Bukhsh and S Pathak ldquoHomeopathic drug discov-ery theory update and methodological aspectrdquo Expert Opinionon Drug Discovery vol 3 no 8 pp 979ndash990 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


of drug that can be identified by specific receptors of thecells as a trigger to turn ldquoonrdquo or ldquooffrdquo some relevant genesinitiating a cascade of gene actions to alter and correct thegene expressions that might have gone wrong during produc-tion of the pathological disorderdisease condition [16 19]Considering that eukaryotic gene expression is an intricateprocess still not completely understood it is difficult withthe knowledge we have at the moment to precisely suggestthe actual molecularmechanisms involved in transmission ofinformation of homeopathic remedies down to the executionlevel for active recovery process [19] For validating thebasis of UHD efficacy and for understanding their molecularmechanism of action further development of basic researchis highly desirable

The microarray technology has been proven to be asuitable high throughput tool to simultaneously analyze alarge number of genestargets associated with the therapeuticeffects of CAM remedies such as herbal drugs [20] Theworking principle for this genomic approach is that thephenotype of a cell including the function and responseto the environment is ultimately determined by its geneexpression profiles Application of such expression analysistools may help to understand and characterize the genes andsignaling pathways involved in CAMmodalities

Microarray analyses have been used to investigate geneexpression variation in human cell lines and animal modelsafter treatment with high dilutions (HDs) and UHDs [21 22]or homeopathic formulations [23] Recently Saha et al [24]used global microarray profiling of genes to demonstrate thatthe expression of certain genes in cancer cells treated withUHDs was significantly different from that of the placebotreated cells

At present microarray profiling of genes to verify theeffects of UHDs on plant systems has never been usedPlant models appear to be useful approaches to address basicquestions on homeopathy research as they make it possibleto overcome some of the drawbacks encountered in clinicaltrials such as placebo effect ethical issues duration and highcosts [25ndash27] The in vitro growth of wheat coleoptiles is oneof the first models adopted to investigate the effects of UHDs[28] and has become a classic test system for basic research inthis field [29] In previous experiments [30 31] we observed acurative effect of arsenic trioxide (As

2O3) at the 45th decimal

dilutiondynamization level on wheat seedling growth Thistreatment applied after having poisoned the seeds with a sub-lethal dose of As

2O3 induced a significant increase in shoot

length compared to poisoned seeds grown in distilled waterIn the present study we reproduced our original exper-

iment [30] in order to investigate the global effects ofultrahigh diluted As

2O3treatments on wheat coleoptiles

gene expression through microarray analyses (Gene ChipsAffymetrix CA) The overall goal was to test the hypothesiswhether wheat coleoptiles grown from As

2O3poisoned

seeds showed different significant gene expression profilesafter the application of ultrahigh diluted As

2O3compared

to water (placebo) At the present state of our knowledgethis is the first time that the microarray profiling approachis being used on plant organisms for gene expression stud-ies on UHDs This might throw additional insight into

the molecular mechanism involved in the biological action ofultrahigh diluted homeopathic drugs

2 Materials and Methods

21 Plant Material and Experimental Treatments Seeds ofthe common wheat (Triticum aestivum L) cultivar ldquoPandasrdquo(CGS sementi Italy) were selected for integrity uniform sizeshape and colour and used to carry out the study

Part of the wheat seeds was pretreated (poisoned) byimmersion for 30min in a 5mM As

2O3watery solution

(As2O3 puriss pa 9995ndash10005 Sigma-Aldrich St Louis

MO USA H2O pa Merck Darmstadt Germany) rinsed in

tap water for 60min dried at ambient air until 12 humiditywas attained and stored in the darkness until use Thissublethal poisoning protocol was selected on the basis ofprevious range and time exposure-finding trials [30] Controlseedswere pretreated in the sameway but with distilledwaterinstead of As

2O35mM

Each seed (poisoned and unpoisoned) was fixed on theupper part of a filter paper (Perfecte 2-extrarapid CordenonsPordenone Italy) by a piece of clay (020 plusmn 005 g) Thefilter paper was inserted into a transparent polyethylenebag (12 times 20 cm) which in turn was placed into a largerblack cardboard envelope covering the roots but not theshoots [30]This technique ensured the growth of shoots androots in natural light and darkness respectively 32mL ofdistilled water or As

2O345x (freshly prepared and supplied

by Laboratoires Boiron Sainte-Foy-les-Lyon France) wasadded in each polyethylene bag during seed growth [3031] As

2O345x treatment (containing a theoretical dose of

As2O31 times 10minus47M) was prepared according to the European

Pharmacopoeia through serial dilutions (in distilled water)at decimal scale (indicated as x) starting from a mother tinc-ture (As

2O3001M) and intercalated by mechanical shaking

(dynamization process) The distilled water used for controland serial dilutions was from the same batch The followingexperimental groups were set up (1) control (denoted by C +0) seeds grown in distilled water without prior treatment (2)treated control (denoted by C + T) seeds grown in As

2O3

45x without prior treatment (3) poisoned (denoted by P +0) seeds pretreated with a sublethal dose of As

2O3and

subsequently grown in distilled water (4) poisoned-treated(denoted by P + T) seeds pretreated with a sublethal doseof As2O3and subsequently grown in As

2O345x A total of

80 cardboard envelopes (20 per each of the 4 experimentalgroups) were fixed to a wooden support and seeds weregrown in the laboratory at a temperature of 20 plusmn 1∘C indaytime assuring a natural day-night rhythm for 7 daysTreatments (water or As

2O345x) were letter-coded (blind

protocol) by a person not involved in the experimentationand were poured into the polyethylene bags according to acompletely randomized design Seven independent replicatedexperiments were carried out for a total of 140 seedlingsgrown for each group out of 560 processed seedlings

The rationale behind poisoning seeds with arsenic andthen growing seedlings in ultrahigh diluted arsenic comesfrom one of the basic tenets of complementary medicinesusing UHDs the principle of ldquolike cures likerdquo In other words




2O345x)


2O3for the stress





2O3was provided


2O345x








































2O345x































Table 2 Continued






























Table 2 Continued














P + 0

P + T versus C + 0

versus C + 0

601 146 232


423 169 474









DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing


Hormonemetabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynthesis

Wall

Cell

Organization

Transport

Developmepp nt

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1








DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1











DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1












DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing

Secondary metabo

Hormmetab


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynth

Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1




2O345x
























2O345x







2O3in two














2O3









4 Conclusions











Acknowledgments


References

































2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2O345x)


2O3for the stress





2O3was provided


2O345x








































2O345x































Table 2 Continued






























Table 2 Continued














P + 0

P + T versus C + 0

versus C + 0

601 146 232


423 169 474









DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing


Hormonemetabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynthesis

Wall

Cell

Organization

Transport

Developmepp nt

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1








DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1











DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1












DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing

Secondary metabo

Hormmetab


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynth

Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1




2O345x
























2O345x







2O3in two














2O3









4 Conclusions











Acknowledgments


References

































2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































2O345x































Table 2 Continued






























Table 2 Continued














P + 0

P + T versus C + 0

versus C + 0

601 146 232


423 169 474









DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing


Hormonemetabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynthesis

Wall

Cell

Organization

Transport

Developmepp nt

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1








DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1











DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1












DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing

Secondary metabo

Hormmetab


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynth

Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1




2O345x
























2O345x







2O3in two














2O3









4 Conclusions











Acknowledgments


References

































2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































Table 2 Continued






























Table 2 Continued














P + 0

P + T versus C + 0

versus C + 0

601 146 232


423 169 474









DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing


Hormonemetabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynthesis

Wall

Cell

Organization

Transport

Developmepp nt

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1








DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1











DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1












DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing

Secondary metabo

Hormmetab


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynth

Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1




2O345x
























2O345x







2O3in two














2O3









4 Conclusions











Acknowledgments


References

































2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 2 Continued














P + 0

P + T versus C + 0

versus C + 0

601 146 232


423 169 474









DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing


Hormonemetabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynthesis

Wall

Cell

Organization

Transport

Developmepp nt

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1








DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1











DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1












DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing

Secondary metabo

Hormmetab


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynth

Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1




2O345x
























2O345x







2O3in two














2O3









4 Conclusions











Acknowledgments


References

































2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing


Hormonemetabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynthesis

Wall

Cell

Organization

Transport

Developmepp nt

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1








DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1











DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1












DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing

Secondary metabo

Hormmetab


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynth

Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1




2O345x
























2O345x







2O3in two














2O3









4 Conclusions











Acknowledgments


References

































2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1











DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1












DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing

Secondary metabo

Hormmetab


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynth

Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1




2O345x
























2O345x







2O3in two














2O3









4 Conclusions











Acknowledgments


References

































2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

minus3

minus2

minus1

0

3

2

1












DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing

Secondary metabo

Hormmetab


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynth

Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1




2O345x
























2O345x







2O3in two














2O3









4 Conclusions











Acknowledgments


References

































2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DNA

SynthesisSynthesis

Regulation

Processing


Hormone metabolism


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes


Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

Abiotic



Repair

DNA

SynthesisSynthesis

Regulation

Processing

Secondary metabo

Hormmetab


Fermentation

Aminoacids

TCA transformation

Lipid

Cofactors

Nucleotides

RNAProtein

Synthesis

Modification

Degradation

Targeting

Enzymes

Photosynth

Wall

Cell

Organization

Transport

Development

Regulation

Stress

Biotic

AbioticAbiotic


Unknown


Repair

minus3

minus2

minus1

0

3

2

1




2O345x
























2O345x







2O3in two














2O3









4 Conclusions











Acknowledgments


References

































2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























2O345x







2O3in two














2O3









4 Conclusions











Acknowledgments


References

































2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























2O3









4 Conclusions











Acknowledgments


References

































2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


References

































2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























2O3high dilution

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Transcriptome Profiling of Wheat Seedlings following Treatment with Ultrahigh Diluted Arsenic...

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Transcript of Transcriptome Profiling of Wheat Seedlings following Treatment with Ultrahigh Diluted Arsenic...