The Spermine Phosphate-Bound Cyclooctaoxygen Sodium Epigenetic Shell of Euchromatin DNA is Destroyed by the Epigenetic Poison Glyphosate

Oxygen exists in two gaseous and six solid allotropic modifications. An additional allotropic modification of oxygen, the cyclooctaoxygen, was predicted to exist in 1990. The first synthesis and characterization of cyclooctaoxygen as its sodium crown complex, isolated in the form of three cytosine nucleoside hydrochloride complexes, was reported in 2016. Cyclooctaoxygen sodium was synthesized from atmospheric oxygen, or catalase effect-generated oxygen, under catalysis of cytosine nucleosides and either ninhydrin or eukaryotic low-molecular weight RNA. The cationic cyclooctaoxygen sodium complex was shown to bind RNA and DNA, to associate with single-stranded DNA and spermine phosphate, and to be essentially non-toxic to cultured mammalian cells at 0.1–1.0 mM concentration. We postulated that cyclooctaoxygen is formed in most eukaryotic cells from dihydrogen peroxide in a catalase reaction catalysed by cytidine and RNA. A molecular biological model was deduced for a first epigenetic shell of eukaryotic euchromatin. This model incorporates an epigenetic explanation for the interactions of the essential micronutrient selenium (as selenite) with eukaryotic euchromatin. The sperminium phosphate/cyclooctaoxygen sodium complex is calculated to cover the actively transcribed regions (2.6%) of bovine lymphocyte interphase genome. Cyclooctaoxygen seems to be naturally absent in hypoxia-induced highly condensed chromatin, taken as a model for eukaryotic metaphase/anaphase/early telophase mitotic chromatin. We hence propose that the cyclooctaoxygen sodium-bridged spermine phosphate and selenite coverage serves as an epigenetic shell of actively transcribed gene regions in eukaryotic ‘open’ euchromatin DNA. The total herbicide glyphosate (ROUNDUP) and its metabolite (aminomethyl)phosphonic acid (AMPA) are proved to represent ‘epigenetic poisons’, since they both selectively destroy the cyclooctaoxygen sodium complex. This definition is of reason, since the destruction of cyclooctaoxygen is sufficient to bring the protection shield of human euchromatin into collateral epigenetic collapse.


The Isolation of Two Cyclooctaoxygen Complexes NC and dNC
In an endeavor to gain new antiviral substances, the reported reaction [11] of the RNA nucleoside cytidine with ninhydrin on reflux was re-examined.Instead of cytidine, cytidine hydrochloride (cytidine × HCl) was utilized.The reported reaction [11] did not proceed, instead a crystalline material NC could be isolated which gave not the elemental analysis of cytidine × HCl.It could be substantiated that NC contained an inert material not being salt (NaCl), since the Fourier transform infrared (FT-IR) spectrum of NC differed from that of cytidine × HCl.According to elemental analysis this inert material could account for one O2 and a quarter of NaCl pro one cytidine × HCl.In consequence, the formula was multiplied fourfold and this resulted in an oxygen 8-ring, cyclo-O8 (Figure 2A), coordinated to one Na + (Figure 2B).
The interpretation of the electrospray ionization mass spectrometry (ESI-MS) spectrum of NC actually proved the inclusion of cyclo-O8-Na + in NC.Since in the proton nuclear magnetic resonance ( 1 H-NMR) spectrum of NC, in comparison to the 1 H-NMR reference spectrum of cytidine × HCl, the differentially affected resonances were the 4-NH2, the 3-NH + , and the H-5 protons of the protonated cytidine, it was assumed [5] that the points of coordination between cyclo-O8-Na + and cytidine × HCl are the two 4-NH2 hydrogens and one non-Na + -coordinated (free) oxygen of cyclo-O8-Na + .Consequently, a formula for NC was elaborated: cytidine hydrochloride -aqua(chloro)(octoxocane- 4 O 1 ,O 3 ,O 5 ,O 7 )sodium (4:1) (Figure 4A) [5].

Catalase Assay of NC and Candida utilis RNA -Biomimetic Synthesis of RC -Structure Proof for Cyclooctaoxygen
It was questioned if cyclo-O8-Na + could be produced in biomimetic reactions, and it was considered that in the two ninhydrin reactions atmospheric oxygen was the source of the oxygen atoms in cyclo-O8.Our interest concentrated on oxygen formation by possible catalase effects under physiological conditions.The catalase effect is the disproportionation of dihydrogen peroxide (H2O2) into oxygen and water: 2 H2O2 → O2 + 2 H2O.As a catalyst RNA was selected, since RNA can exhibit enzymatic (ribozyme) activities in vivo [12,13].The selected eukaryotic RNA was Candida utilis anamorph yeast low-molecular weight RNA.This RNA consists of transfer RNAs (tRNAs) and the C. utilis 5S ribosomal RNA (rRNA) [5].As a result it was discovered that NC catalysed oxygen formation from H2O2 (catalase effect) weakly in presence of NaHCO3, and strongly in presence of both C. utilis RNA and NaHCO3 [5].Interestingly, NC could be fully substituted by cytidine × HCl.Multiple controls assured that oxygen neither was produced spontaneously, nor from any other relevant combination of the utilized reagents.Taken together, the nucleoside cytidine, not cyclo-O8-Na + , was responsible for the catalase activity expressed in presence of H2O2 and C. utilis RNA under biomimetic conditions.It was decided to exactly scale-up (21-fold) the catalase assay protocol starting with cytidine × HCl and C. utilis RNA to detect any cyclo-O8-Na + formation under biomimetic conditions.From this preparation a cyclo-O8-Na + -containing crystalline material RC could be isolated which gave not the elemental analysis of cytidine × HCl.If the C. utilis RNA was omitted, no product RC could be isolated, only cytidine × HCl.Based on 1 H-NMR spectroscopy and FT-IR spectroscopy of RC, a formula for RC could be constructed: cytidine hydrochloride --chloro(-hydroxy)bis(octoxocane- 4 O 1 ,O 3 ,O 5 ,O 7 )disodium (1:2) (Figure 4C) [5].

Binding of NC to Candida utilis RNA
In view of the biomimetic generation of the cyclo-O8-Na + -containing coordination complex RC, the question arose if cyclo-O8-Na + could bind to nucleic acids, because of the mere electrostatic attraction of the cyclo-O8-Na + cation towards the negatively charged phosphate backbone of RNA and DNA.For this purpose thin-layer chromatographic mobility shift assays [14] were applied on specific nucleic acids and the cyclo-O8-Na + contained in NC.Firstly, the affinity of the cyclo-O8-Na + towards C. utilis lowmolecular weight RNA was investigated [5].It was found that the cyclo-O8-Na + contained in NC retained the chromatographic shift of C. utilis 5S rRNA, but not the chromatographic shift of C. utilis tRNAs.Interestingly, since work conditions were not human skin ribonuclease (RNase)-free, the RNase A digestion products of C. utilis 5S rRNA were separated chromatographically [5].These dinucleotide 2',3'cyclic phosphates (products of RNase A digestion) result from human skin RNase 7-mediated digestion of C. utilis 5S rRNA [5].The structures of these dinucleotides can be deduced, since RNase 7 belongs to the RNase A superfamily [5].The cyclo-O8-Na + contained in NC bound strongly to these dinucleotide 2',3'-cyclic phosphates, since their chromatographic shifts were significantly retarded.Controls were included to differentiate the sole binding of cytidine × HCl to the RNA targets by Watson-Crick base pairing [15] from the indicative cyclo-O8-Na + plus cytidine × HCl binding to the RNA targets.

Binding of NC to Salmon Testes Single-Stranded DNA and Spermine Phosphate
Accordingly, the affinity of the cyclo-O8-Na + contained in NC towards salmon testes singlestranded deoxyribonucleic acid [ssDNA, generated by sonication of salmon genomic double-stranded DNA (dsDNA); extracted after sonication by phenol-chloroform method and precipitated with ethanol; the sonication shears the genomic dsDNA to produce ssDNA fragments in the range of 587 to 831 bp] was investigated (Figure 6) [5].It was found that the cyclo-O8-Na + contained in NC retained the chromatographic shift of cytidine × HCl complexed to ssDNA (Figure 6).As control served cytidine × HCl complexed to ssDNA.The affinity of the cyclo-O8-Na + contained in NC towards salmon testes ssDNA in absence and presence of spermine × 1 ⅓ (sodium dihydrogen phosphate) × 9 H2O was investigated [5].It was found that the spermine × 1 ⅓ (sodium dihydrogen phosphate) × 9 H2O changed the chromatographic shift of the cytidine × HCl in NC-complexed ssDNA.As controls served cytidine × HCl complexed to ssDNA in absence and presence of cyclo-O8-Na + , and cytidine × HCl complexed to ssDNA in presence of spermine × 1 ⅓ (sodium dihydrogen phosphate) × 9 H2O [5].Taken together, cyclo-O8-Na + contained in NC had the ability to bind to RNA dinucleotide 2',3'-cyclic phosphates, eukaryotic 5S rRNA, eukaryotic ssDNA, and to construct a ternary complex with spermine phosphate and eukaryotic ssDNA.

In Vitro Biological Effects of NC and dNC on Cultured Mammalian Cells
The in vitro biological effects of NC and dNC on the growth of cultured cells, freshly explanted human primary (human peripheral blood mononuclear cells, PBM cells), immortalized T-lymphoblastic (CCRF-CEM) and monkey kidney normal epithelial (Vero), were investigated [5].NC and dNC were non-toxic to PBM cells, but stimulated the growth of CCRF-CEM cells.This pointed to a catalase effect exerted by NC and dNC, since CCRF-CEM cells are extremely sensitive to H2O2 [5], and scavenging of H2O2 by 'catalase factors' is CCRF-CEM cell growth rate-limiting [5].Since NC was more active as a growth stimulant for CCRF-CEM cells than dNC, the responsible 'catalase factors' should be the nucleoside hydrochlorides, not the equimolar cyclo-O8-Na + -content in NC and dNC.NC and dNC exhibited no significant in vitro antiviral activities against the retro-transcribing human immunodeficiency type 1 and hepatitis B viruses (HIV-1 strain LAI and HBV subtype ayw) [5].NC showed no significant in vitro inhibiting activity versus the replication of influenza A (H1N1 and H5N1) and chikungunya (strain S-27) viruses, and no significant in vitro inhibiting activity on Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV strain Erasmus Medical Center/2012) replication [5].In summary, cyclo-O8-Na + is, contrary to expectation, essentially non-toxic to human cells, and cytidine in conjunction with RNA acts as a catalyst in producing cyclo-O8-Na + from ubiquitous [5] H2O2 through a catalase reaction in cultured human cells.
A logically deduced catalytic 'rolling-circle' mechanism for the AMPA-catalysed degradation of cyclo-O8 is hence proposed (Figure 8C).AMPA exhibits three acid dissociation constants: pKa1 = 0.9 (phosphonic acid, 1 st ), pKa2 = 5.6 (phosphonic acid, 2 nd ), pKa3 = 10.2 (primary ammonium R-NH3 + ) [23].Therefore, AMPA is fully (zwitter)ionized at physiological pH 7.4.One anionic oxygen of the phosphonate group binds to the sodium cation in cyclo-O8-Na + , the other anionic phosphonate oxygen splits the cycloocytooxygen ring creating a phosphonate-esterified nonaoxidanide which is stabilized by ionic binding to the primary ammonium cation of AMPA.The phosphonate-esterified nonaoxidanide eliminates four oxygen O2 molecules by a 'rolling-circle' cascade, in reversal of the proposed [5] synthesis of cyclooctaoxygen, yielding AMPA and Na + .This would be clearly a catalytic mechanism, since AMPA is regenerated in the catalytic cycle.Hence AMPA is able to destroy many cyclo-O8-Na + complexes without being consumed itself.
To exclude that the destruction of cyclo-O8-Na + by AMPA is an artifact, it was tested if AMPA reduces (decolorizes) iodine in near equimolar mixture (Figure 7).In all variations tested, including a blank control, AMPA was not oxidized by iodine, and, in turn, did not reduce (decolorize) iodine molecules.Therefore, the AMPA-catalysed destruction of cyclo-O8 was selective, and not a mere reduction of the tetraiodide I4 2-[(I-I-I-I) 2-]-contained iodine unit in the [(cyclo-O8-Na + )2(I4 2-)]-amylose complex.
A logically deduced catalytic 'rolling-circle' mechanism for the glyphosate-catalysed degradation of cyclo-O8 is hence proposed (Figure 10).One anionic oxygen of the phosphonate group binds to the sodium cation in cyclo-O8-Na + , the other anionic phosphonate oxygen splits the cycloocytooxygen ring creating a phosphonate-esterified nonaoxidanide which is stabilized by ionic binding to the secondary ammonium cation of glyphosate.The phosphonate-esterified nonaoxidanide eliminates four oxygen O2 molecules by a 'rolling-circle' cascade, yielding glyphosate and Na + in a catalytic mechanism.Glyphosate is regenerated in the catalytic cycle.Glyphosate is able to destroy many cyclo-O8-Na + complexes without being consumed itself.
To exclude that the destruction of cyclo-O8-Na + by glyphosate is an artifact, it was tested if glyphosate, or glyphosate-Na, reduces (decolorizes) iodine in near equimolar mixture (Figure 9).Glyphosate was not oxidized by iodine, and, in turn, did not reduce (decolorize) iodine molecules.Therefore, the glyphosate-catalysed destruction of cyclo-O8 was selective.To confirm the general nature of the color assay, it was extended to the cyclo-O8-Na + contained in NC (1 mol cyclo-O8-Na + pro 4 mol cytidine × HCl) [5].The reddish violet [(cyclo-O8-Na + )2(I4 2-)]-amylose complex was indeed formed from NC (Figure 9), but much more slowly (> 10 h) than from RC.
Our findings have important consequences for the epigenetics [35] of eukaryotic in vivo DNA.We suggested [5] a model for a first epigenetic shell of in vivo DNA (Figure 3), based on the observed complexation of cyclo-O8-Na + and spermine phosphate to ssDNA.In our model (Figure 3A) the phosphate backbone of ssDNA binds one cyclo-O8-Na + pro three nucleotides, and this binary complex binds one spermine monophosphate to form a ternary epigenetic core of DNA.The monohydrogen phosphate bridges the cyclo-O8-Na + with the sperminium cation, and the cyclo-O8-Na + has an inverted alternating orientation (Figure 3A).Interestingly, the sperminium tetracation cannot bind alone to DNA in this model, since the distances [d (N 1 ,N 4 ) = 490 pm; d (N 4 ,N 9 ) = 620 pm; d (N 1 ,N 12 ) = 1,600 pm] between the four ammonium nitrogens do not fit the intrastrand phosphate-phosphate distance of dsDNA (B-DNA: dØ = 700 pm [36,37]; A-DNA: dØ = 590-600 pm [37][38][39][40]; Z-DNA: dØ = 590 pm (step pCp), dØ = 600 pm (step pGp) [40]).Therefore, it is quite remarkable that in our model for the first epigenetic shell of in vivo DNA (Figure 3A) a repeating unit is formed from cyclo-O8-Na + and spermine phosphate that perfectly fits both the triplet nature of the genetic code [41] and the repeating distance of the phosphate anion backbone of DNA.
Evidence for the correctness of this model results from the published investigation of spermine distribution in bovine lymphocytes [42].The theoretical intracellular concentration of the sperminium phosphate/cyclo-O8-Na + complex required to cover all triplets of the dsDNA genome in a blood lymphocyte of Bos taurus was calculated as 13.81 mM (see Methods 4.2.) [5].The actual concentration of spermine was measured as 1.57 ± 0.12 (mM ± s.d.) [42].Therefore, the genomic dsDNA coverage of B. taurus genome can be calculated as 2.62 ± 0.50 (% ± s.d.) (see Methods 4.2.) [5], since one unit of sperminium phosphate/cyclo-O8-Na + complex is assumed to cover three nucleotides.A good correlation was obtained when this value was compared to the proportion of protein-coding exons in B. taurus genome which was calculated as 2.58% (see Methods 4.2.).For comparison, the human genome contains 2.69% protein-coding exons (see Methods 4.6.).This pointed to complete coverage of actively transcribed gene regions in B. taurus interphase genome by the sperminium phosphate/cyclo-O8-Na + complex.Since spermine binds more strongly to GC-rich dsDNA (pBR322 plasmid) [42], it can be assumed that the sperminium phosphate/cyclo-O8-Na + complex binds preferentially to epigenetic, non-5-methylated CpG island hotspots [5] and is involved in epigenetic gene regulation [5].
In view of the important findings of Kirmes et al. [4] we were not aware of in 2015, that an interaction of eukaryotic chromatin DNA structure with atmospheric oxygen partial pressure takes place, we have to correct now previous postulations [5].We concluded that the cyclooctaoxygen sodiumbridged spermine phosphate epigenetic shell is confined to both interphase relaxed euchromatin and mitotic condensed chromatin [5].Since, under switching to hypoxic conditions eukaryotic cell chromatin gets highly condensed [4], accompanied by redistribution of the polyamine pool to the nucleus [4], the cyclooctaoxygen sodium-bridged spermine phosphate epigenetic shell can only be restricted to actively transcribed gene regions of eukaryotic 'open' euchromatin, excluding occupation of condensed chromatin.Hypoxia should largely prevent metabolic formation of cyclooctaoxygen.Both under hypoxic conditions and in the metaphase of mitosis, where spermine synthesis is highest [43], coincident with an extraordinary high condensation grade (15,000-20,000-fold) of metaphase chromatin [44], no or few cyclooctaoxygen should be involved in covering the highly condensed chromatin DNA.Here no or few discrimination between eu-and heterochromatin is made, and all eukaryotic chromatin DNA is complexed with spermine tetracation and spermidine trication (and, at small proportions, with putrescine and cadaverine dications).This is supported by the published concentration of spermine in the metaphase chromatin of eukaryotic HeLa S3 cells [45].The content of spermine in HeLa S3 cell metaphase chromatin was calculated as 135.9 ± 16.1 pmol spermine/82.84zmol dsDNA, and 116.1 ± 11.8 pmol spermidine/82.84 zmol dsDNA (see Methods 4.3.)[5,45].This corresponds to 1.64 × 10 9 molecules spermine pro one HeLa S3 cell dsDNA genome, and 1.40 × 10 9 molecules spermidine pro one HeLa S3 cell dsDNA genome.Since one spermine molecule is assumed to cover six base pairs (in the pure spermine form of A-DNA duplex [46] and Z-DNA duplex [47]), and one spermidine molecule is assumed to cover six base pairs (in the pure spermidine form of Z-DNA duplex [48,49]), this corresponds to a genomic coverage of 50.4% by the spermine tetracation, and of 43.0% by the spermidine trication.This accounts for 93.4% polyamine occupation of HeLa S3 cell dsDNA highly condensed metaphase chromatin by spermine and spermidine, calculated for six base pairs/polyamine unit.As one spermine molecule, in one special occasion, was found to cover four base pairs of an unique B-DNA [50], these values could be anticipated as being lower, since chromosomal DNA is predominantly in the B-DNA form.
Indirect control (we must here regretfully correct our postulations stated in [5], concerning this calculation) for this in vitro result is the published elemental phosphorus content [w (P) in mmol/kg dry weight] in female Mus musculus strain C3H/HeJ cryptal enterocytic mitotic (late anaphase/early telophase) chromatin [51].The obtained in vivo value corresponds to a genomic dsDNA coverage (calculated for 6 bp/polyamine) of 102.1% (100% coverage is 5.88 bp/polyamine), and a nuclear RNA coverage (calculated for 6 nucleotides/polyamine) of 159.5%, by the spermine tetracation and spermidine trication (spermine/spermidine ratio 0.85) (see Methods 4.4.).This strongly points to a function of polyamine occupation for nuclear RNA, assuming 100% coverage as 3.76 nucleotides/polyamine molecule.These results, both for dsDNA and nuclear RNA [hnRNA with pre-mRNAs, snRNA, snoRNA, RNase P, RNase MRP, various ncRNAs (lncRNA) and other nuclear RNAs] [52], are a logic consequence of the maximal condensation grade peaking in late anaphase/early telophase mammalian chromatin [53].
In summary, this reflects the high mitotic chromatin condensation grade and is confirming the results with hypoxia-induced chromatin condensation under coinciding polyamine pool nuclear translocation [4].Interestingly, spermine and spermidine induced B-DNA to Z-DNA transition at epigenetic, non-5-methylated CpG island hotspots of prokaryotic plasmid DNA (pBR322 derivative) [54], but, in contrast, stabilized and condensed prokaryotic chromosomal B-DNA [55].Z-DNA was found to be formed at CpG island transcriptional hotspots [56,57].Regions near the transcription start site frequently contain sequence motifs favorable for forming Z-DNA, and formation of Z-DNA near the promoter region stimulates transcription [57].All these observations point to the correctness of our model that the cyclooctaoxygen sodium-bridged spermine phosphate epigenetic shell is restricted to actively transcribed This is substantiated by the precise calculation of the apparent acid dissociation constant of the human genome DNA (see Methods 4.5.).The apparent (effective) pK′a,HG (25 °C) = 7.18 of the haploid human genome was calculated (Figure 12A) according to the method of Katchalsky & Gillis [58], as based on the theoretical considerations of Kuhn & Kuhn [59].The hypothetical intranuclear pHDNA = 1.66, mediated by H. sapiens haploid interphase genome dsDNA without any neutralizing shell, can be calculated (Figure 12B) (see Methods 4.6.).Assuming one spermine molecule covering four base pairs (single occupation) of B-DNA [50], and correcting for actively transcribed gene regions of H. sapiens genome, the hypothetical intranuclear micro-pH [60] surrounding H. sapiens haploid interphase euchromatin when covered (single quartet occupation) by the spermine tetracation alone can be calculated pHDNA/spermine = 7.43 (see Methods 4.6.).For the diploid dsDNA genome, after completed S phase during interphase, the pHDNA/spermine is identical.The theoretical intranuclear micro-pH surrounding H. sapiens haploid interphase euchromatin when covered (double triplet occupation) by the sperminium phosphate/cyclooctaoxygen sodium complex can be calculated pHDNA/shell = 7.42 (see Methods 4.7.).For the diploid dsDNA genome, after completed S phase during interphase, the pHDNA/shell is identical.
We also elaborated a model for selenium (as hydrogen selenite, HSeO3 -, at physiological pH 7.4) protection of DNA (Figure 3B) [5].Selenium, the element of the moon [61], was discovered by Jöns Jacob Berzelius (1779-1848) in 1817 and was named by him in honor of the Greek goddess of the moon Selene (σελήνη) [62].Selenium is essential to mammalian physiology at nutritional levels, but supraphysiological intake of selenium is known to be toxic for mammals [5,7].Sodium selenite (Na2SeO3), as hydrogen selenite HSeO3 -at pH 7.0 (selenious acid H2SeO3: pKa1 = 2.62, pKa2 = 8.32 [63]), binds to calf thymus genomic B-DNA at pH 7.0 [64], and to Saccharomyces cerevisiae A-RNA at pH 7.0 [65].Selenium has the ability to protect DNA from noxious influences (oxidative stress, radiation, cytotoxic agents) [5], and is essential to genomic stability [5,7,66,67], but the exact molecular biological basis for these phenomena is unknown.If in our model of a first epigenetic shell of in vivo DNA (Figure 3A) the monohydrogen phosphate is replaced by hydrogen selenite (Figure 3B), an epigenetic explanation for the interaction of selenium with eukaryotic in vivo DNA could be given.This model may account for, at least some of, the well-known bimodal, protective and toxic, in vivo effects exerted by selenium onto mammalian physiology [5,7].A moderate substitution pattern of hydrogen selenite for monohydrogen phosphate would be essential, but if the displacement ratio HSeO3 -/HPO4 2-exceeds a certain tolerance level, the epigenetic equilibrium should collapse.The extraordinary high, both acute and chronic, mammalian toxicity of sodium selenite (Na2SeO3) [68] should be due, at least in part, to direct detrimental effects of supraphysiological levels of hydrogen selenite HSeO3 -on mammalian chromosomal DNA integrity and regulation of genome expression.In fact, Na2SeO3 is a violent poison with a lethal dose 50% (LD50, orally in rats, 7 mg/kg [69]), being lower than the LD50 of sodium cyanide (NaCN) (LD50, orally in rats, 15 mg/kg [70]).
Assuming an essential biological function for the cyclooctaoxygen sodium-bridged spermine phosphate and selenite epigenetic shell, we searched for substances able to selectively destroy this epigenetic protection structure, and tested the total herbicide glyphosate, N-(phosphonomethyl)glycine (ROUNDUP®, Monsanto), and its major environmental metabolite (aminomethyl)phosphonic acid (AMPA) [71] on the cyclo-O8-Na + complex contained in RC.Glyphosate was chosen because it represents the top selling total herbicide worldwide [72], and RC was selected because of its highest molar cyclo-O8-Na + content in the complex series NC, dNC, and RC (Figure 4) [5].Glyphosate and AMPA show chemical properties which might predispose them for destruction of cyclooctaoxygen in general.Glyphosate and AMPA are very hydrophilic and amphoteric, and their phosphonate moieties could be suitable to interact with cyclo-O8-Na + .We could show unequivocally that glyphosate and AMPA indeed selectively destroy the cyclo-O8-Na + complex contained in RC (see Methods 4.8., 4.9.and 4.10.)(Figure 7, 8, 9 and 10).We therefore conclude that glyphosate and the major environmental glyphosate metabolite AMPA [71] also destroy the cyclooctaoxygen sodium-bridged spermine phosphate and selenite epigenetic shell of human euchromatin, because destruction of cyclooctaoxygen is sufficient to bring this essential protection shield of human euchromatin into collateral epigenetic collapse.
To get support for the selectivity of AMPA as an epigenetic poison, the affinity of AMPA towards human mitochondrial -aminobutyric acid transaminase (ABAT) [25], and to wild-type human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) [26], was determined (see Methods 4.11.and 4.12.).ABAT represents also a -alanine transaminase [25], and both enzymes were selected because of the structural similarity between AMPA and -alanine/L-alanine.AMPA showed essentially no affinity to ABAT, but was very showly catabolized by AGT (Figure 11A).This latter result is of interest, since the product of the half-transamination of AMPA by AGT is phosphonoformaldehyde which can be oxidized (peroxisomal glycolate oxidase, cytoplasmic lactate dehydrogenase [73]) to phosphonoformic acid (phosphonoformate, forscarnet) (Figure 11B).Foscarnet represents a well-known inhibitor of mammalian [74,75] and viral [74,75] DNA-dependent DNA polymerases.Eukaryotic DNA polymerase  is crucially involved in chromosome maintenance, DNA repair and recombination, transcriptional silencing, checkpoint activation, and telomere length maintenance [76].Mammalian DNA polymerase  is potently inhibited by foscarnet [74,75].Therefore, the low-affinity half-transamination of AMPA by AGT, the ratelimiting step leading to foscarnet, could negatively influence human chromosome maintenance, DNA damage repair, and telomere length preservation, mediated by the AMPA catabolite foscarnet inhibition of DNA polymerase .This enzymatic catabolism provides an additional, minor mechanism of destabilization and impairment of eukaryotic chromosomal DNA indirectly induced by the environmental glyphosate metabolite AMPA.
The stock solutions were incubated at room temperature (RT, ϑ = 14.0 °C) for 30 min, then at elevated temperature (ϑ = 25.2 °C) for 2 min, after which time the first photograph series (Figure 7) was taken.The solutions were then incubated at elevated temperature for 48 min, after which time the second photograph (Figure 7) series was taken.The solutions were further incubated at RT for 14 h.Afterwards, both RC + KI (3) and RC + KI + AMPA (4) were mixed with 1,000 µl deuterated chloroform (CDCl3), and extracted by shaking.Concomitantly, RC + KI + starch (5) and RC + KI + starch + AMPA (6) were both mixed with 8 mg of solid L-ascorbic acid (concentration of L-ascorbic acid in solution 22.7118 mM).The solutions were succeedingly incubated at elevated temperature for 15 min, after which time the third photograph series (Figure 7) was taken.

Control Color Assay for Potential Reduction of Elemental Iodine by the Glyphosate Metabolite (Aminomethyl)phosphonic Acid
Stock preparations were: blank without AMPA (1,000 µl H2O), AMPA stock solution (34 mg AMPA in 1,000 µl H2O), and two iodine stock solutions [each 80 mg elemental iodine in 2,000 µl 90% (v/v) aqueous ethanol].The stock preparations were incubated at room temperature (RT, ϑ = 14.0 °C) for 15 min with occasional shaking, then at elevated temperature (ϑ = 25.2 °C) for 15 min, until the stock preparations were nearly dissolved (AMPA full, iodine not fully).Then the AMPA stock solution was injected into the first iodine stock solution (it results solution A), no decoloration resulted.The H2O blank was injected into the second iodine stock solution (it results solution B), no decoloration resulted.The concentrations in solution were now: AMPA, 102.0653 mM; iodine as I2, 105.0655 mM.
After incubation at elevated temperature for 15 min, into both solutions 1,000 µl of water were injected, crystallization of elemental iodine followed, and no decoloration resulted.The first photograph series (Figure 7) was taken after 25 min incubation at elevated temperature.After incubation at elevated temperature for 15 min, 2,000 µl of 90% (v/v) aqueous ethanol were injected in both A and B. The mixtures were shaken, the iodine dissolved to give clear deep brown solutions, and no decoloration resulted.After 5 min incubation at elevated temperature the second photograph (Figure 7) series was taken.Both A and B did not show any further change at RT during 24 h observation.
A color assay for cyclo-O8-Na + contained in NC was accordingly performed.The preparation was: NC + KI + starch (25 mg NC + 43 mg KI + 18 mg starch in 1,000 µl H2O).The concentrations in solution were: NC, 18.2452 mM (with cyclo-O8-Na + , 18.2452 mM); KI, 259.0361 mM.The preparation was incubated at elevated temperature for 10 h, after which time the first photograph (Figure 9) series was taken.The preparation was further incubated at room temperature (RT, ϑ = 13.7 °C) for 40 h, after which time the second photograph series (Figure 9) was taken.
To investigate whether AMPA is a substrate for human mitochondrial GABA transaminase, experiments were performed with commercially available recombinant human GABA transaminase according to the procedure of Schor et al. [25] with some modifications.Incubations with 500 nmol AMPA, in an assay volume of 120 µl did not show activity of GABA transaminase towards AMPA, while the control assay using 500 nmol 15 N-GABA as substrate did resulted in the formation of the expected enzyme product.Subsequent inhibition experiments, with co-incubations of fixed amounts (500 nmol) of 15 N-GABA with increasing amounts of AMPA (0-2000 nmol), revealed that AMPA did not act as an inhibitor of the GABA transaminase-catalysed reaction of 15 N-GABA to succinic semialdehyde.These combined results strongly suggest that AMPA is not a substrate for human GABA transaminase.

Enzyme Assay of the Glyphosate Metabolite (Aminomethyl)phosphonic Acid with Human Wild-Type Alanine:Glyoxylate Aminotransferase
Recombinant human alanine:glyoxylate aminotransferase was expressed in E. coli and purified as described [91].The enzyme at 5 µM concentration was incubated with 100 mM AMPA at 25 °C in 100 mM potassium phosphate buffer pH 7.4.At various times (1, 2, 5, 22 h), aliquots were withdrawn and the reaction was stopped by adding trichloroacetic acid 10% (v/v).The total amount of PLP and pyridoxamine 5'-phosphate (PMP) was determined by HPLC analysis as previously described (Figure 11A) [92].

Conclusions
We allow us the profound conclusions that sperminium phosphate/cyclo-O8-Na + coverage of nucleic acids is essential for eukaryotic gene regulation, and, in conjunction with selenite, protects and stabilizes gene-rich 'open' chromatin euchromatic DNA [5].These postulations [5] would account for a long-sought molecular explanation of the essential, but 'mysterious' function of the polyamine spermine in eukaryotes [6].Spermine is found only in eukaryotes, with some exceptions, and prokaryotes rely mostly on putrescine and spermidine [6,93].The essentiality of spermine for humans is exemplified by the Snyder-Robinson X-linked mental retardation syndrome [94] caused by missense mutations in the human spermine synthase gene, leading to mental retardation, generalised seizures, absent speech, inability to stand, and other severe defects [94].One can speculate that at the transition from prokaryotic to eukaryotic life the sperminium phosphate/cyclo-O8-Na + complex resulted as a consequence from the combined accumulation of atmospheric oxygen and prokaryotic RNA, since the evolution of spermine synthases from prokaryotic spermidine synthase was proposed [93] as co-occurring with the onset of proto-eukaryotic life.
An improved and corrected molecular biological model is proposed for a first epigenetic shell of eukaryotic euchromatin.This model incorporates an epigenetic explanation for the interactions of the essential micronutrient selenium (as selenite) with eukaryotic euchromatin.The sperminium phosphate/cyclooctaoxygen sodium complex was calculated to cover the actively transcribed regions (2.6%) of bovine lymphocyte interphase genome dsDNA (double occupation).The polyamine (spermine/spermidine ratio 1.17) coverage of HeLa S3 cell metaphase chromatin dsDNA was calculated as 93.4% (single occupation).In murine cryptal enterocytic mitotic (late anaphase/early telophase) chromatin the obtained in vivo value corresponds to complete genomic coverage (single occupation), and to comprehensive and extensive nuclear RNA coverage, by the spermine tetracation and spermidine trication (spermine/spermidine ratio 0.85).Because cyclooctaoxygen seems to be naturally absent in hypoxia-induced highly condensed chromatin [4], we hence propose a model [95] for the cyclooctaoxygen sodium-bridged spermine phosphate (and selenite) epigenetic shell of actively transcribed gene regions in eukaryotic 'open' chromatin DNA (Figure 13).Furthermore, a working model is tabulated in summary for the selective cell cycle-dependent epigenetic occupation of eukaryotic DNA (Table 1).
What may be the overall biological significance, and pathophysiological implication, of this selective epigenetic shell?During transcription of actively transcribed gene regions in eukaryotic 'open' chromatin the double helix must be unwound by DNA helicases [96] and the strands must be separated to enable access to DNA-dependent RNA polymerases I, II [97,98], and III.This creates intermediate DNA single-strand regions which are prone to chemical structure damage by multiple noxious impacts like reactive oxygen species (ROS) [3] and mutagens [99].The selective cyclooctaoxygen sodium-bridged spermine phosphate (and selenite) epigenetic occupation of these sensitive single-stranded stretches could serve as an intrinsic protection against chemically-induced structural damage.This would be a logic explanation for the selective nature of the separate occupation of both DNA strands, consequently retained when strands are separated for transcription of mRNA.
But what is the chemical, obviously evolutionary conserved, genomic necessity for the DNA single-strand protection by cyclooctaoxygen sodium-bridged spermine complexes?Since spontaneous deamination, ROS, chemical mutagens, and UV light do damage both dsDNA and ssDNA regions [100][101][102], albeit ssDNA with higher propensity than dsDNA [100,101], the immediate benefit must be based in another origin.Intriguing seem to be pH effects, since spermine is a strong base, and the major pHrelated damage to DNA is depurination creating apurinic sites at low pH [103][104][105], with a four times higher reaction rate for ssDNA than for dsDNA [103,105].
The formation of kinetin (N 6 -furfuryl-9H-adenine) from DNA is known [106].It should be emphasized that kinetin is not contained in native mammalian DNA, contrary to misleading claims [107,108], but is formed only during DNA damage.A mechanism for the kinetin formation in, or from, DNA was proposed [107,108], but it seems not to be conclusive in chemical reason, since furfural does not react with the adenine 6-NH2 group under condensation to a Schiff base [106].We therefore propose a chemical mechanistic deduced logical scheme [109][110][111][112] for the generation of kinetin from DNA by proton catalysis (kinetin-generating "base flip", KGBF) (Figure 14), based on proton-catalysed depurination and subsequent inverted adenine 6-NH2 N-glycosylation [109,110], in consequence leaving back a DNA single-strand break.It is proposed that the cyclooctaoxygen sodium-bridged spermine phosphate epigenetic shell protects ssDNA from low pH-induced depurination, including, in part, generation of kinetin by KGBF.This is substantiated by the precise calculation of the apparent acid dissociation constant of the human genome DNA (see Methods 4.5.).
We therefore conclude that the sperminium phosphate/cyclooctaoxygen sodium complex serves to protect ssDNA from nucleic acid-mediated intrinsic low intranuclear micro-pH-induced depurination, including KGBF, creating apurinic sites and concomitant DNA single-strand breaks at eukaryotic genome regions engaged in active transcription.The precisely calculated intranuclear micro-pH gain, obtained by sperminium phosphate/cyclooctaoxygen sodium complexation of B-DNA individual strands, is essentially the same as the intranuclear micro-pH gain for condensed B-DNA strand-overarchingly covered by sperminium tetracations.
In conclusion, it is logically obvious that any chemical agent, biochemical precursor (selenium) deficiency, and/or physical circumstance compromising the sperminium phosphate/selenitecyclooctaoxygen sodium complexation will inevitably lead to a severe disturbation of eukaryotic genome integrity, to an increased mutation rate, and to genomic DNA single-strand breaks caused by KGBF.This is, in part, proved by the Snyder-Robinson X-linked mental retardation syndrome [94], characterized by a defect in spermine synthesis, leading to nearly complete loss of the polyamine spermine.We therefore investigated chemical agents selectively destroying the epigenetic shell of eukaryotic euchromatin, found a candidate molecule, and, hence, wish to define it as an 'epigenetic poison'.The total herbicide glyphosate, N-(phosphonomethyl)glycine (ROUNDUP®, Monsanto), and its major environmental metabolite (aminomethyl)phosphonic acid (AMPA) [71] were found, rather unequivocally, to selectively destroy the cyclo-O8-Na + complex contained in RC (Figure 7, 8, 9 and 10).Glyphosate and AMPA came into focus because (i) glyphosate represents the top selling total herbicide worldwide [72], (ii) their chemical structure (phosphonate + amine) and properties (strongly hydrophilic and acidic) seemed to enable them to interact with cyclooctaoxygen sodium, (iii) glyphosate and ROUNDUP® are suspected to damage DNA and cause cancer in humans [113], and (iv) AMPA is already widely distributed in global ecosystems like (surface) water [114].
We allow us to conclude on basis of our, rather unequivocal, findings that glyphosate, ROUNDUP® and AMPA are major examples of slow-acting, insidious 'epigenetic poisons', (i) slowly eroding and detoriating human, animal and plant genomic integrity, (ii) rattening human, animal and plant inborne protection of hereditary information against mutation, and (iii) disturbing the processing of

Figure 1 .
Figure 1.Parts of the original publication [1] from 1677 by Antoni van Leeuwenhoek with the description of the first light microscopic observation of crystalline spermine phosphate in human semen.(A) The title page 1040.(B) Page 1042 with fig.A showing the characteristic crystalline shape [2] of spermine × 2 H3PO4 × 6 H2O [2].The last paragraph including fig.A is read in New Latin: "Et sum praedicta materia paucillum temporis steterat, in ea observabantur trilaterales figurae ab utraque parte in aculeum desinentes, quibusdam longitudo minutissimae arenae, aliquae aliquantulum majores, ut fig. A. Praetera, adeo nitidae ac pellucidae, ac si crystallinae fuissent.".English transcription: "And I mentioned the matter which stood for a short time, in which trilateral figures were observed from both sides ending in a sting, some in length of minute grains, some a little larger, as fig. A. Moreover, so sleek and translucent, as if it were crystalline.".

Figure 10 .Figure 11 .
Figure 10.A logically deduced catalytic 'rolling-circle' mechanism for the (fully ionized) glyphosatecatalysed degradation of cyclo-O8-Na + .The cycloocytooxygen ring is split to a phosphonate-esterified nonaoxidanide which is stabilized by ionic binding to the secondary ammonium cation of glyphosate (and complexation of the sodium cation).The phosphonate-esterified nonaoxidanide eliminates four oxygen O2 molecules by a 'rolling-circle' cascade, regenerating glyphosate.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 25 January 2017 doi:10.20944/preprints201701.0086.v2 human
, animal and plant genetic information by transcription.It is hence inevitable for us to define glyphosate, ROUNDUP® and AMPA as a significant threat for human, animal and plant genomic stability, especially for future human generations forced to live under the glyphosate-, ROUNDUP®-and AMPA-induced radiomimetic effects.

Table 1 .
Tabulation of the selective cell cycle-dependent occupation of eukaryotic DNA by epigenetic polyamine shells. 1 ×, single quartet occupation (one polyamine pro both strands); 2 ×, double triplet occupation (one polyamine pro one strand); Chr, chromatin.