Going off the rails with the biomarker paradigm

This 2018 US government rodent study used extreme dosages to achieve its directed goals of demonizing nicotine and extolling the biomarker paradigm:

“This study examined whether adolescent nicotine exposure alters adult hippocampus-dependent learning, involving persistent changes in hippocampal DNA methylation and if choline, a dietary methyl donor, would reverse and mitigate these alterations.

Mice were chronically treated with nicotine (12.6mg/kg/day) starting at post-natal day 23 (pre-adolescent), p38 (late adolescent), or p54 (adult) for 12 days followed by a 30-day period during which they consumed either standard chow or chow supplemented with choline (9g/kg).

Our gene expression analyses support this model and point to two particular genes involved in chromatin remodeling, Smarca2 and Bahcc1. Both Smarca2 and Bahcc1 showed a similar inverse correlation pattern between promoter methylation and gene expression.

Our findings support a role for epigenetic modification of hippocampal chromatin remodeling genes in long-term learning deficits induced by adolescent nicotine and their amelioration by dietary choline supplementation.”


Let’s use the average weight of a US adult male – published by the US Centers for Disease Control as 88.8 kg – to compare the study’s dosages with human equivalents:

  1. Nicotine at “12.6mg/kg/day” x 88.8 kg = 1119 mg. The estimated lower limit of a lethal dose of nicotine has been reported as between 500 and 1000 mg!
  2. Choline at “9g/kg” x 88.8 kg = 799 g. The US National Institutes of Health published the Tolerable Upper Intake Levels for Choline as 3.5 g!!

The human-equivalent dosage of nicotine used in this study would probably kill an adult human. Who knows what exceeding the choline “Tolerable Upper Intake Level” BY 228 TIMES would do to an adult human.

Is the main purpose of animal studies to help humans? Or is it to promote an agenda?


A funding source of this study was National Institute on Drug Abuse (NIDA) Identification of Biomarkers for Nicotine Addiction award (T-DA-1002 MG). Has the biomarker paradigm been institutionalized to the point where research proposals that don’t have biomarkers as goals aren’t funded?

https://www.sciencedirect.com/science/article/pii/S107474271830193X “Choline ameliorates adult learning deficits and reverses epigenetic modification of chromatin remodeling factors related to adolescent nicotine exposure” (not freely available)

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How do memories transfer?

This 2018 Chinese study electronically modeled the brain’s circuits to evaluate memory transfer mechanisms:

“During non-rapid-eye-movement (NREM) sleep, thalamo-cortical spindles and hippocampal sharp wave-ripples have been implicated in declarative memory consolidation. Evidence suggests that long-term memory consolidation is coordinated by the generation of:

  • Hierarchically nested hippocampal ripples (100-250 Hz),
  • Thalamo-cortical spindles (7-15 Hz), and
  • Cortical slow oscillations (<1 Hz)

enabling memory transfer from the hippocampus to the cortex.

Consolidation has also been demonstrated in other brain tasks, such as:

  • In the acquisition of motor skills, where there is a shift from activity in prefrontal cortex to premotor, posterior parietal, and cerebellar structures; and
  • In the transfer of conscious to unconscious tasks, where activity in initial unskilled tasks and activity in skilled performance are located in different regions, the so-called ‘scaffolding-storage’ framework.

By separating a neural circuit into a feedforward chain of gating populations and a second chain coupled to the gating chain (graded chain), graded information (i.e. information encoded in firing rate amplitudes) may be faithfully propagated and processed as it flows through the circuit. The neural populations in the gating chain generate pulses, which push populations in the graded chain above threshold, thus allowing information to flow in the graded chain.

In this paper, we will describe how a set of previously learned synapses may in turn be copied to another module with a pulse-gated transmission paradigm that operates internally to the circuit and is independent of the learning process.”


The study has neither been peer-reviewed, nor have the mechanisms been tested in living beings.

https://www.biorxiv.org/content/early/2018/07/27/351114 “A Mechanism for Synaptic Copy between Neural Circuits”

Epigenetic variations in metabolism

This 2018 German review was comprehensive for its subject, epigenetic control of variation and stochasticity in metabolic disease. I’ll focus on one aspect, phenotypic variation:

“Phenotypic [Mendelian] variation can result both from gain- and loss-of-function mutations. Because of the extreme interconnectivity of cell regulatory networks, even at the cellular level, predicting the impact of a sequence variant is difficult as the resultant variation acts:

  • In the context of all other variants and
  • Their potential additive, synergistic and antagonistic interactions.

This phenomenon is known as epistasis.

∼98.5% of our genome is non-protein-coding: it is pervasively transcribed, and its transcripts can support regulatory function. Among the best functionally characterized non-coding RNAs (ncRNAs) arising from these sequences are microRNAs (miRNAs)

Environmental [non-Mendelian] variation or ‘stimuli’ occurring during critical windows of susceptibility can elicit lifelong alterations in an individual’s phenotype. Intergenerational metabolic reprogramming [in fruit flies] results from global alterations in chromatin state integrity, particularly from reduced H3K27me3 and H3K9me3 [histone] domains.

The broad variation of fingerprints in humans is thought to depend to a large degree on stochastic variation in mechanical forces. These clear examples of inducible multi-stable or stochastic variation highlight how little we know about the landscape of potential phenotypic variation itself.

Consensus estimates of heritability for obesity and T2D are ∼70% and ∼35% respectively. The remaining, unexplained component is known to involve gene–environment interactions as well as non-Mendelian players.”


Although the above graphic displays transgenerational inheritance for humans, the reviewers didn’t cite any human studies that adequately demonstrated causes for and effects of transgenerational epigenetic inheritance.

I’ve read the cited Swedish and Dutch studies. Their designs, methods, and “correlate with” / “was associated with” results didn’t provide incontrovertible evidence from the F0 great-grandparents, F1 grandparents, F2 parents, and F3 children. It’s necessary to thoroughly study each generation to confirm definitive transgenerational epigenetic inheritance causes and effects.

As noted in How to hijack science: Ignore its intent and focus on the 0.0001%, there aren’t any such published studies to cite. Researchers urgently need to do this human research, and stop using these poor substitutes [1] to pretend there are already adequately evidenced transgenerational epigenetic inheritance human results.

I downgraded the review for treating research of this and other subjects as faits accomplis. It’s opposite ends of the evidential spectrum to state “how little we know about the landscape of potential phenotypic variation,” and in the same review, speciously extrapolate animal experiments into putative human results.

https://www.sciencedirect.com/science/article/pii/S2212877818301984 “Epigenetic control of variation and stochasticity in metabolic disease”


[1] As an example of the poor substitutes for evidence, a researcher referred me to the 2013 “Transgenerational effects of prenatal exposure to the 1944–45 Dutch famine” which is freely available at https://obgyn.onlinelibrary.wiley.com/doi/full/10.1111/1471-0528.12136 as a study finding human transgenerational epigenetic inheritance.

The methods section showed:

  • The study’s non-statistical data was almost all self-reported by a self-selected sample of the F2 grandchildren, average age 37.
  • No detailed physical measurements or samples were taken of them, or of their F1 parents, or of their F0 grandparents, all of which are required as baselines for any transgenerational epigenetic inheritance.
  • No detailed physical measurements or samples were taken of their F3 children, which is the generation that may provide evidence for transgenerational findings if the previous generations also have detailed physical baselines.

The study’s researchers drew enough participants (360) such that their statistics package allowed them to impute and assume into existence a LOT of data. But the scientific method constrained them to make factual statements of what the evidence actually showed. They admitted:

“In conclusion, we did not find a transgenerational effect of prenatal famine exposure on the health of grandchildren in this study.”

Yet this study is somehow cited for evidence of human transgenerational epigenetically inherited causes and effects.

A dietary supplement that trains the innate immune system

This 2018 Netherlands review topic was the long-term epigenetic programming of the innate immune system:

“Immunological memory has been classically described for the adaptive immune system, in which naive B and T lymphocytes develop antigen-specific, long-lasting memory cells after encountering a new antigen.

Immunological memory is not an exclusive trait of lymphocytes. The function of cells from the innate immune system, such as monocytes, macrophages, dendritic cells, and NK cells, is also influenced by the contact with different stimuli, undergoing functional reprogramming.

β-glucan, the prototypical trained immunity-inducing agonist:

  • Modulates hematopoietic stem and progenitor cells, influencing the behavior and responsiveness of peripheral myeloid cells;
  • Leads to a shift of cellular metabolism from oxidative phosphorylation toward aerobic glycolysis.

Analysis of transcriptional data from macrophages stimulated with β-glucan revealed that the cholesterol synthesis pathway is highly up-regulated in trained immunity. A follow-up of this study showed that the activation of the cholesterol synthesis pathway, but not its synthesis itself, is crucial for innate memory. In agreement with this, the inhibition of cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan.

β-glucan-induced changes in trimethylation of histone 3 lysine 4 (H3K4me3) and acetylation of histone 3 lysine 27 (H3K27ac) in human monocytes 7 days after the first stimulation in vitro were associated with a switch to glycolysis, suggesting a deep, long lasting reprogramming of the cells.

Inducers of cellular reprogramming such as β-glucan have shown potential as a treatment or adjuvant for osteosarcoma, influenza, or skin lesions, among others.”


β-glucan is available as a supplement at $.17 a day and through eating oatmeal and barley. Why don’t cereal companies advertise the immune system benefits of their products?

https://jlb.onlinelibrary.wiley.com/doi/pdf/10.1002/JLB.MR0318-104R “Long-term reprogramming of the innate immune system” (not freely available)

A mid-year selection of epigenetic topics

Here are the most popular of the 65 posts I’ve made so far in 2018, starting from the earliest:

The pain societies instill into children

DNA methylation and childhood adversity

Epigenetic mechanisms of muscle memory

Sex-specific impacts of childhood trauma

Sleep and adult brain neurogenesis

This dietary supplement is better for depression symptoms than placebo

The epigenetic clock theory of aging

A flying human tethered to a monkey

Immune memory in the brain

The lack of oxygen’s epigenetic effects on a fetus

Addictive behavior and epigenetic DNA methylation

This 2018 McGill paper reviewed findings from animal and human studies on the relationships between drug-seeking behavior and epigenetic DNA methylation:

“Although there is an increasing line of evidence from preclinical models of addiction, there are only a few human studies that systematically assessed DNA methylation in addiction. Most of the studies were done on small cohorts and focused on one or a few candidate genes, except in the case of alcohol use where larger studies have been carried out.

A long line of evidence suggests that abnormal patterns of gene expression occur in brain regions related to drug addiction such as the nucleus accumbens, prefrontal cortex, amygdala, and the ventral tegmental area.

Using the “incubation of craving” model in rats trained to self-administer cocaine, and treated with either SAM or RG108, the genome-wide DNA methylation and gene expression landscape in the nucleus accumbens after short (1 day) and long (30 days) abstinence periods and the effects of epigenetic treatments were delineated. The main findings are:

  • A long incubation period results in robust changes in methylation;
  • Direct accumbal infusion of SAM that is paired with a “cue” after long incubation times increases drug-seeking behavior,
  • Whereas a single treatment with RG108 decreases this behavior.

Importantly, the effects of these single administrations of a DNA methylation inhibitor remain stable for 30 more days. These data suggest that DNA methylation might be mediating the impact of “incubation” on the craving phenotype and that this phenotype could be reprogrammed by a DNA demethylation agent.”


The subject has a large scope, and a narrow aspect was presented in this paper. Rodent research by one of the coauthors that was cited, Chronic pain causes epigenetic changes in the brain and immune system, provided some relevant details.

The review covered neither human dimensions of the impacts of unfulfilled needs nor investigations of exactly what pain may impel human drug-seeking behavior. The “Implications for Diagnostic and Therapeutics” were largely at the molecular level.

https://www.sciencedirect.com/science/article/pii/S1877117318300164 “The Role of DNA Methylation in Drug Addiction: Implications for Diagnostic and Therapeutics” (not freely available)

Melatonin and depression

This 2018 Polish review subject was the relationship between melatonin and depression:

“Although melatonin has been known about and referred to for almost 50 years, the relationship between melatonin and depression is still not clear. In this review, we summarize current knowledge about the genetic and epigenetic regulation of enzymes involved in melatonin synthesis and metabolism as potential features of depression pathophysiology and treatment.

Melatonin has an antidepressant effect by:

  • Maintaining the body’s circadian rhythm,
  • Regulating the pattern of expression of the clock genes in the suprachiasmatic nucleus (SCN) and
  • Modifying the key genes of serotoninergic neurotransmission that are linked with a depressive mood.

Light input causes the release of γ-aminobutyric acid (GABA) by the SCN, and the inhibitory signal is transmitted to the pineal gland to inhibit melatonin production.

Melatonin is produced via the metabolism of serotonin in two steps which are catalyzed by serotonin N-acetyltransferase (SNAT) and acetylserotonin-O-methyltransferase (ASMT). Serotonin, SNAT, and ASMT are key melatonin level regulation factors.

Both melatonin and serotonin are synthesized from the same amino acid, tryptophan. People on a high tryptophan diet (>10 mg/kg body weight per day) have a significantly lower level of depressive symptoms, irritation, and anxiety than people on a low tryptophan diet (<5 mg/kg body weight per day).

To our knowledge, there are only 2 studies in the literature that characterize mRNA expression of ASMT in the peripheral blood of recurrent DD [depressive disorders]. [They] have demonstrated the reduced mRNA expression of ASMT in patients with depression and cognitive impairment. Surprisingly, these studies, despite promising results, have not been replicated. Moreover, no analysis of other melatonin related-genes as potential biomarkers of depression has been provided.

The main monoamine hypothesis of the pathophysiology of depression indicates that depression is induced by a change in the level of ≥1 monoamines such as serotonin, noradrenaline, and dopamine. The evidence for the serotonergic theory is an observation that antidepressants such as tricyclic antidepressants, selective serotonin reuptake inhibitors, and noradrenaline reuptake inhibitors increase the level of serotonin in the brain.

We focus on serotonin as a neurotransmitter which is a precursor of melatonin synthesis. In a depressed patient, serotonin synthesis is impaired and the poor precursor availability may prevent the formation of an adequate amount of melatonin. However, only a few studies have analyzed the relationship between serotonin and melatonin levels and the correlation with the blood serum.”


At eight cents a day ($.04 for women) melatonin is a cheap and effective supplement.

I hadn’t considered possible antidepressant effects until reading this review. More human studies are needed.

https://www.karger.com/Article/Pdf/489470 “Pathophysiology of Depression: Molecular Regulation of Melatonin Homeostasis – Current Status” (not freely available)