Biochim Biophys Acta-Gen Subj.2016 Apr;1860(4):785–794. doi: 10.1016/j.bbagen.2016.01.022.
A new oxygen modification cyclooctaoxygen binds to nucleic acids as sodium crown complex
Andreas J. Kesel a,*, Craig W. Day b, Catherine M. Montero c,d, Raymond F. Schinazi c,d
a Chammünsterstr. 47, D-81827 München, Bavaria/Bayern, Germany.
b Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Science, Utah State University, Logan, Utah 84322, United States.
c Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322, United States.
d Veterans Affairs Medical Center, Decatur, Georgia 30033, United States.
* Correspondence to: A.J. Kesel, Chammünsterstr. 47, München 81827, Germany.
E-mail address: firstname.lastname@example.org (A.J. Kesel). Telephone: +49 (0)89-453 64 500.
Background: Oxygen exists in two gaseous and six solid allotropic modifications. An additional allotropic modification of oxygen, the cyclooctaoxygen, was predicted to exist in 1990.
Methods: Cyclooctaoxygen sodium was synthesized in vitro from atmospheric oxygen, or catalase effect-generated oxygen, under catalysis of cytosine nucleosides and either ninhydrin or eukaryotic low-molecular weight RNA. Thin-layer chromatographic mobility shift assays were applied on specific nucleic acids and the cyclooctaoxygen sodium complex.
Results: We report the first synthesis and characterization of cyclooctaoxygen as its sodium crown complex, isolated in the form of three cytosine nucleoside hydrochloride complexes. The cationic cyclooctaoxygen sodium complex is shown to bind to nucleic acids (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.
Conclusions: We postulate that cyclooctaoxygen is formed in most eukaryotic cells in vivo from dihydrogen peroxide in a catalase reaction catalysed by cytidine and RNA. A molecular biological model is deduced for a first epigenetic shell of eukaryotic in vivo DNA. This model incorporates an epigenetic explanation for the interactions of the essential micronutrient selenium (as selenite) with eukaryotic in vivo DNA.
General significance: Since the sperminium phosphate/cyclooctaoxygen sodium complex is calculated to cover the active regions (2.6%) of bovine lymphocyte interphase genome, and 12.4% of murine enterocyte mitotic chromatin, we propose that the sperminium phosphate/cyclooctaoxygen sodium complex coverage of nucleic acids is essential to eukaryotic gene regulation and promoted proto-eukaryotic evolution.
PMID: 26825775. PMCID: PMC4780752.
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.
In 1677 Antoni van Leeuwenhoek discovered  the characteristic crystals of spermine phosphate (spermine × 2 H3PO4 × 6 H2O)  in matured native human semen. Since then, there was collected conclusive evidence that human chromosomal DNA is closely associated with spermine phosphate . The interaction of oxygen species with DNA has until recently only being focused on oxidative DNA damage and its pathophysiological consequences. In 2015 Kirmes et al. reported an unprecedented interaction of eukaryotic chromatin DNA structure with atmospheric oxygen partial pressure . Under switching to hypoxic conditions (1% O2, 5% CO2, 94% N2) the murine cardiomyocyte HL-1 cell chromatin rendered itself highly condensed, accompanied by redistribution of the polyamine pool (mainly spermine and spermidine) to the nucleus . In 2016 Kesel et al. showed  that eukaryotic single-stranded DNA (ssDNA) binds a new allotropic form of oxygen, the cyclooctaoxygen (cyclo-O8), in form of its sodium (Na+) complex (cyclo-O8-Na+), especially when in coordination to spermine phosphate (sperminium phosphate) . A model for a logically resulting first epigenetic shell of eukaryotic DNA in vivo was proposed (Figure 1) . Also a partial substitution of the sperminium (C10H30N44+)-bound monohydrogen phosphate (HPO42–) anions by hydrogen selenite (HSeO3–) anions was postulated during these investigations , thereby providing an explanation for the well-known, but ‘mysterious’ , augmenting effects of the polyamine spermine  and the essential micronutrient selenium on eukaryotic genome integrity and chromosomal DNA stability .
Figure 1. Molecular modeling [ACD/Chem Sketch version 12.01 with integrated ACD/3D Viewer (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada), Mercury 3.1 version 3.1.1 (The Cambridge Crystallographic Data Centre, Cambridge, United Kingdom)] of the postulated  first epigenetic shell of euchromatic in vivo DNA, as exemplified for a single-stranded hexanucleotide, introducing a molecular biological model for sperminium phosphate/cyclo-O8-Na+/ssDNA and sperminium selenite/cyclo-O8-Na+/ssDNA interactions. (A) The molecular model of the single-stranded hexanucleotide d(ApApApApApAp) liganded with cyclo-O8-Na+ and sperminium phosphate. (B) The molecular model of d(ApApApApApAp) liganded with cyclo-O8-Na+ and sperminium selenite. Element color codings for (A) and (B): gray, carbon; white, hydrogen; blue, nitrogen; red, oxygen; purple, phosphorus; green, sodium; yellow, selenium.
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 . These postulations  would account for a long-sought molecular explanation of the essential, but ‘mysterious’ function of the polyamine spermine in eukaryotes . Spermine is found only in eukaryotes, with some exceptions, and prokaryotes rely mostly on putrescine and spermidine . The essentiality of spermine for humans is exemplified by the Snyder–Robinson X-linked mental retardation syndrome  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 . 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  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 , we hence propose a model for the cyclooctaoxygen sodium-bridged spermine phosphate (and selenite) epigenetic shell of actively transcribed gene regions in eukaryotic ‘open’ chromatin DNA (Figure 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 and the strands must be separated to enable access to DNA-dependent RNA polymerases I, II, 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) and mutagens. 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.
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, 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/selenite–cyclooctaoxygen 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. This is, in part, proved by the Snyder–Robinson X-linked mental retardation syndrome , 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)  were found, rather unequivocally, to selectively destroy the cyclo-O8-Na+ complex contained in RC (Figures 2, 3 and 4). Glyphosate and AMPA came into focus because (i) glyphosate represents the top selling total herbicide worldwide , (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 , and (iv) AMPA is already widely distributed in global ecosystems like (surface) water .
Figure 2. Color assays for cyclo-O8-Na+ contained in RC, for the destruction of cyclo-O8-Na+ by the glyphosate metabolite (aminomethyl)phosphonic acid (AMPA) (left, 1–6), and for the potential reduction of elemental iodine by AMPA (right, A and B). Solutions (left, 1–6) were: KI (1), KI + starch (2), RC + KI (3), RC + KI + AMPA (4), RC + KI + starch (5), and RC + KI + starch + AMPA (6). The concentrations in solution were: RC, 16.95 mM (with cyclo-O8-Na+, 67.79 mM); KI, 156.63 mM; AMPA, 99.06 mM. The solutions were incubated at two room temperatures for prolonged time. Afterwards (left, bottom row), both RC + KI (3) and RC + KI + AMPA (4) were extracted with deuterated chloroform (bottom phase), and (left, bottom row) both RC + KI + starch (5) and RC + KI + starch + AMPA (6) were treated with L-ascorbic acid. Legend: left, top row (1–6) = first photograph series; left, middle row (1–6) = second photograph series; left, bottom row (1–6) = third photograph series; right, top row (first photograph series): (A) AMPA (76.55 mM) + iodine (as I2, 78.80 mM), (B) iodine (as I2, 78.80 mM); right, bottom row (second photograph series): (A) AMPA (51.03 mM) + iodine (as I2, 52.53 mM), (B) iodine (as I2, 52.53 mM).
Figure 3. Color assays for the destruction of cyclo-O8-Na+ contained in RC by glyphosate and ROUNDUP® GRAN (top, 1–5), for the potential reduction of elemental iodine by glyphosate and ROUNDUP® GRAN (bottom, A1, A2, and B), and for cyclo-O8-Na+ contained in NC (bottom, NC1 and NC2). Solutions (top, 1–5) were: RC + KI + starch (1), RC + KI + starch + glyphosate (free acid) (2), RC + KI + starch + ROUNDUP® GRAN (3), RC + KI + glyphosate (free acid) (4), and RC + KI + ROUNDUP® GRAN (5). The concentrations in solution were: RC, 16.95 mM (with cyclo-O8-Na+, 67.79 mM); KI, 171.69 mM; glyphosate, 100.55 mM; glyphosate-Na, 108.15 mM. The solutions were incubated at two room temperatures for prolonged time. Legend (bottom): (A1) glyphosate (free acid, 102.52 mM) + iodine (as I2, 98.50 mM), (A2) ROUNDUP® GRAN (glyphosate-Na, 102.77 mM) + iodine (as I2, 98.50 mM), (B) iodine (as I2, 98.50 mM), (NC1, NC2) NC (18.25 mM, with cyclo-O8-Na+, 18.25 mM) + KI (259.04 mM) after 10 h (NC1) and 50 h (NC2) incubation.
Figure 4. A logically deduced catalytic ‘rolling-circle’ mechanism for the (fully ionized) glyphosate-catalysed 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.
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 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.
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Acknowledgements: The authors thank T. Westfeld, E.-M. May, D. Wiegel, W. Wübbolt, O. Meier, R. Sachs, A. Karbach, W. Bergmeier, J. Moldenhauer and H.-J. Hühn (Currenta GmbH & Co. OHG, Leverkusen, Germany) for analytical services. We thank H.J. Jodl for helpful discussions, and Nathan Clyde for performing the RNA virus assays at Utah State University. This work was supported in part by NIH CFAR grant 2P30–AI–50409 (to R.F.S.) and by the Department of Veterans Affairs (to R.F.S.). We are obliged to K. Hecker, K. Meuser and K. Hecker (HEKAtech GmbH, Wegberg, Germany) for expert elemental analyses.
Andreas Johannes Kesel
Chammünsterstr. 47, D-81827 München, Bavaria/Bayern, Germany