An epigenetic clock review by committee

This 2019 worldwide review of epigenetic clocks was a semi-anonymous mishmash of opinions, facts, hypotheses, unwarranted extrapolations, and beliefs. The diversity of viewpoints among the 21 coauthors wasn’t evident.

1. Citations of the coauthors’ works seemed excessive, and they apologized for omissions. However, Challenge 5 was titled “Single-cell analysis of aging changes and disease” and Table 1 “Major biological and analytic issues with epigenetic DNA methylation clocks” had single-cell analysis as the Proposed solution to five of the Significant issues. Yet studies such as High-Resolution Single-Cell DNA Methylation Measurements Reveal Epigenetically Distinct Hematopoietic Stem Cell Subpopulations were unmentioned.

2. Some coauthors semi-anonymously expressed faith that using current flawed methodologies in the future – only more thoroughly, with newer equipment, etc. – would yield better results. If the 21 coauthors were asked their viewpoints of Proposed solutions to the top three Significant issues of epigenetic clocks, what would they emphasize when quoted?

3. Techniques were praised:

“Given the precision with which DNA methylation clock age can be estimated and evolving measures of biological, phenotype-, and disease-related age (e.g., PhenoAge, GrimAge)..”

Exactly why these techniques have at times produced inexplicable results wasn’t examined, though. Two examples:

  • In Reversal of aging and immunosenescent trends, the Levine PhenoAge methodology estimated that the 51-65 year old subjects’ biological ages at the beginning of the study averaged 17.5 years less than their chronological age. Comparing that to the Horvath average biological age of 3.95 years less raised the question: exactly why did PhenoAge show such a large difference?
  • The paper mentioned the GrimAge methodology findings about “smoking-related changes.” But it didn’t explain why the GrimAge methylation findings most closely associated with smoking history also accurately predicted future disease risk with non-smokers.

Eluding explanations for these types of findings didn’t help build confidence in the methodologies.

4. A more readable approach to review by committee could have coauthors – in at least one section – answer discussion questions, as Reversing epigenetic T cell exhaustion did with 18 experts. “DNA methylation aging clocks: challenges and recommendations”

A review of fetal adverse events

This 2019 Australian review subject was fetal adversities:

“Adversity during the perinatal period is a significant risk factor for the development of neurodevelopmental disorders long after the causative event. Despite stemming from a variety of causes, perinatal compromise appears to have similar effects on the developing brain, thereby resulting in behavioural disorders of a similar nature.

These behavioural disorders occur in a sex‐dependent manner, with males affected more by externalizing behaviours such as attention deficit hyperactivity disorder (ADHD) and females by internalizing behaviours such as anxiety. The term ‘perinatal compromise’ serves as an umbrella term for intrauterine growth restriction, maternal immune activation, prenatal stress, early life stress, premature birth, placental dysfunction, and perinatal hypoxia.

The above conditions are associated with imbalanced excitatory-inhibitory pathways resulting from reduced GABAergic signalling. Methylation of the GAD1/GAD67 gene, which encodes the key glutamate‐to‐GABA synthesizing enzyme Glutamate Decarboxylase 1, resulting in increased levels of glutamate is one epigenetic mechanism that may account for a tendency towards excitation in disorders such as ADHD.

The posterior cerebellum’s role in higher executive functioning is becoming well established due to its connections with the prefrontal cortex, association cortices, and limbic system. It is now suggested that disruptions to cerebellar development, which can occur due to late gestation compromises such as preterm birth, can have a major impact on the region of the brain to which it projects.

Activation of the maternal hypothalamic-pituitary adrenal (HPA) axis and placental protection. Psychological stress is perceived by the maternal HPA axis, which stimulates cortisol release from the maternal adrenal gland.

High levels of maternal cortisol are normally prevented from reaching the fetus by the 11β-hydroxysteroid dehydrogenase 2 (HSD11B2) enzyme, which converts cortisol to the much less active cortisone. Under conditions of high maternal stress, this protective mechanism can be overwhelmed, with the gene encoding the enzyme becoming methylated, which reduces its expression allowing cortisol to cross the placenta and reach the fetus.”

The reviewers extrapolated many animal study findings to humans, although most of their own work was with guinea pigs. The “suggest” and “may” qualifiers were used often – 22 and 37 times, respectively. More frequent use of the “appears,” “hypothesize,” “propose,” and “possible” terms was justified.

As a result, many reviewed items such as the above graphic and caption should be viewed as hypothetical for humans rather than reflecting solid evidence from quality human studies.

The reviewers focused on the prenatal (before birth) period more than the perinatal (last trimester of pregnancy to one month after birth) period. There were fewer mentions of birth and early infancy adversities. “Perinatal compromise contributes to programming of GABAergic and Glutamatergic systems leading to long-term effects on offspring behaviour” (not freely available)

May you be the hero who solves your own problems

This 2019 Germany/US review subject was the failure of psychotherapy and pharmacotherapy:

“Each mental disorder raises its own host of issues. However, recent evidence across multiple meta-analyses on key mental disorders provides an overarching picture of limited benefits for both psychotherapy and pharmacotherapy.

Some differences for specific disorders are not strong enough to weaken the overall impression that a dead end has been reached in the treatment of mental disorders. For this reason, a paradigm shift seems to be required.”

Investigate the above linked Primal Therapy category to figure out what you could do for yourself. Follow the below review link for reasons to avoid treatments that waste your one precious life. “Toward a paradigm shift in treatment and research of mental disorders”

Transgenerational epigenetic inheritance of thyroid hormone sensitivity

My 500th curation is a 2019 Portuguese human study of Azorean islanders:

“This study demonstrates a transgenerational epigenetic inheritance in humans produced by exposure to high TH [thyroid hormone] in fetal life, in the absence of maternal influences secondary to thyrotoxicosis. The inheritance is along the male line.

The present work took advantage of the relatively frequent occurrence of fetal exposure to high TH levels in the Azorean island of São Miguel. This is the consequence of a missense mutation in the THRB gene causing the amino-acid replacement R243Q, resulting in reduced affinity of the TH receptor beta (TRβ) for TH and thus RTHβ.

Its origin has been traced to a couple who lived at the end of the 19th century. F0 represented the third generation and F3 the sixth and seventh generation descendant.”

These researchers provided the first adequately evidenced human transgenerational epigenetic inheritance study! However, the lead sentence in its Abstract wasn’t correct:

“Evidence for transgenerational epigenetic inheritance in humans is still controversial, given the requirement to demonstrate persistence of the phenotype across three generations.”

Although found in this study, there is no “requirement to demonstrate persistence of the phenotype.” Observing the same phenotype in each generation is NOT required for human transgenerational epigenetic inheritance to exist!

Animal transgenerational studies have shown that epigenetic inheritance mechanisms may both express different phenotypes for each generation:

and entirely skip a phenotype in one or more generations!

  • Transgenerational pathological traits induced by prenatal immune activation found a F2 and F3 generation phenotype of impaired sociability, abnormal fear expression and behavioral despair – effects that weren’t present in the F1 offspring;
  • The transgenerational impact of Roundup exposure “Found negligible impacts of glyphosate on the directly exposed F0 generation, or F1 generation offspring pathology. In contrast, dramatic increases in pathologies in the F2 generation grand-offspring, and F3 transgenerational great-grand-offspring were observed.” (a disease phenotype similarly skipped the first offspring generation);
  • Epigenetic transgenerational inheritance mechanisms that lead to prostate disease “There was also no increase in prostate histopathology in the directly exposed F1 or F2 generation.” (a prostate disease phenotype skipped the first two male offspring generations before it was observed in the F3 male offspring); and
  • Epigenetic transgenerational inheritance of ovarian disease “There was no increase in ovarian disease in direct fetal exposed F1 or germline exposed F2 generation. The F3 generation can have disease while the F1 and F2 generations do not, due to this difference in the molecular mechanisms involved.” (an ovarian disease phenotype similarly skipped the first two female offspring generations before it was observed in the F3 female offspring).

Details of epigenetic inheritance mechanisms were provided in Another important transgenerational epigenetic inheritance study. Mechanisms from fetal exposure to the fungicide vinclozolin were compared with mechanisms from fetal DDT exposure, and summarized as:

The fetal exposure initiates a developmental cascade of aberrant epigenetic programming, and does NOT simply induce a specific number of DMRs [DNA methylation regions] that are maintained throughout development.

I emailed references to the studies in the first five above curations to the current study’s corresponding coauthor. They replied “What is the mechanism for the transgenerational inheritance you describe?” and my reply included a link to the sixth curation’s study.

Are there still other transgenerational epigenetically inherited effects due to fetal exposure to high thyroid hormone levels? “Reduced Sensitivity to Thyroid Hormone as a Transgenerational Epigenetic Marker Transmitted Along the Human Male Line”

Preliminary findings from a senolytics clinical trial

This 2019 US human clinical trial reported preliminary results:

Senescent cells, which can release factors that cause inflammation and dysfunction, the senescence-associated secretory phenotype (SASP), accumulate with ageing and at etiological sites in multiple chronic diseases. Senolytics, including the combination of Dasatinib and Quercetin (D + Q), selectively eliminate senescent cells by transiently disabling pro-survival networks that defend them against their own apoptotic environment.

Since the target of senolytics is senescent cells, these drugs do not need to be continuously present in the circulation in the same way as drugs whose mechanism of action is to occupy a receptor, modulate an enzyme, or act on a particular biochemical pathway, at least in mice. Intermittently administering D + Q effectively circumvents any potential off-target effects due to continuous receptor occupancy or modulation of an enzyme or biochemical pathway.

To test whether intermittent D + Q is effective in targeting senescent cells in humans, we administered a single 3 day course of oral D + Q and assayed senescent cell abundance 11 days after the last dose in subjects with DKD [diabetic kidney disease], the most common cause of end-stage kidney failure and which is characterized by increased senescent cell burden.

In this interim report of findings, we found the single brief course of D + Q:

  • Attenuated adipose tissue and skin senescent cell burden,
  • Decreased resulting adipose tissue macrophage accumulation,
  • Enhanced adipocyte progenitor replicative potential, and
  • Reduced key circulating SASP factors.”


“In adipose tissue D + Q significantly reduced raw numbers of:

  • p16INK4A+ cells by 35%;
  • p21CIP1+ cells by 17%;
  • SAβgal+ cells by 62%;
  • CD68+ macrophages by 28%; and
  • Crown-like structures by 86%.” “Senolytics decrease senescent cells in humans: Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease”

In a referenced 2019 rodent study by many of the same researchers:

“We also found that even Q alone can prevent high fat diet-induced increases in markers of senescence, renal fibrosis, decreases in renal oxygenation, and increased creatinine in mice, although Q alone did not prevent insulin resistance.”

The rodent study’s 50 mg/kg quercetin dosage would be 375% higher than the 1,000 mg quercetin dosage for a 165-pound (75 kg) clinical trial participant. “Targeting senescent cells alleviates obesity‐induced metabolic dysfunction”

Effects of advanced glycation end products on quality of life and lifespan

This 2018 Chinese review concerned advanced glycation end products (AGE) mobility interventions:

“Only a limited number of studies have focused on measuring the effects of low AGEs levels or AGEs inhibitors on mobility, although many observational human studies and in vitro studies have reported the correlation of AGEs with and the contribution of AGEs to mobility, particular in diseases such as:

  • osteoporosis,
  • cartilage degradation,
  • osteoarthritis and
  • sarcopenia.

There is insufficient information from previous animal and human studies for use as a reference to determine the intervention period. Although serum AGEs levels can be easily affected by a lower AGEs diet or AGEs inhibitors, it may take longer to see the changes in certain organs or tissues, as a result of a reduction in AGEs accumulation.”


“Effect of AGEs on apoptosis signalling. AP-1, activator protein 1; ERK, extracellular signal-regulated protein kinases; IGF-I, insulin-like growth factor I; IL-6, interleukin-6; JAK, Janus kinase; JNK, c-Jun N-terminal kinases; MEK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; p38 MAPK, p38 mitogen-activated protein kinase; RAGE, receptor for AGEs; STAT3, signal transducers and activators of transcription 3; TGF-β, transforming growth factor-β”

Citations aren’t validations of the reference’s quality and strength of evidence. This review would have benefited from not citing reviews that contained misrepresentations, such as one mentioned in Wikipedia is a poor source of information on advanced glycation end products (AGEs).

I came across this review as a result of it citing the excellent 2008 rodent study Oral Glycotoxins Determine the Effects of Calorie Restriction on Oxidant Stress, Age-Related Diseases, and Lifespan which found:

“Higher levels of oxidant AGEs in offspring of Reg-F0 dams may be attributable to placental transmission from mothers with high AGE levels. These high intrauterine AGE levels may predispose the offspring to the development of chronic inflammation and diseases in adulthood, such as insulin resistance and diabetes.

Increasing the intake of AGEs in the diet erases the benefits of CR [calorie restriction]. OS [oxidant stress] can be reduced, and healthspan increased, in mice fed a diet that is restricted in the content of AGEs.

The beneficial effects of a CR diet may be partly related to reduced oxidant intake rather than decreased energy intake.” “Role of advanced glycation end products in mobility and considerations in possible dietary and nutritional intervention strategies”

A drug that countered effects of a traumatizing mother

This 2019 US rodent study concerned transmitting poor maternal care to the next generation:

“The quality of parental care received during development profoundly influences an individual’s phenotype, including that of maternal behavior. Infant experiences with a caregiver have lifelong behavioral consequences.

Maternal behavior is a complex behavior requiring the recruitment of multiple brain regions including the nucleus accumbens, bed nucleus of the stria terminalis, ventral tegmental area, prefrontal cortex, amygdala, and medial preoptic area. Dysregulation within this circuitry can lead to altered or impaired maternal responsiveness.

We administered zebularine, a drug known to alter DNA methylation, to dams exposed during infancy to the scarcity-adversity model of low nesting resources, and then characterized the quality of their care towards their offspring.

  1. We replicate that dams with a history of maltreatment mistreat their own offspring.
  2. We show that maltreated-dams treated with zebularine exhibit lower levels of adverse care toward their offspring.
  3. We show that administration of zebularine in control dams (history of nurturing care) enhances levels of adverse care.
  4. We show altered methylation and gene expression in maltreated dams normalized by zebularine.

These findings lend support to the hypothesis that epigenetic alterations resulting from maltreatment causally relate to behavioral outcomes.”

“Maternal behavior is an intergenerational behavior. It is important to establish the neurobiological underpinnings of aberrant maternal behavior and explore treatments that can improve maternal behavior to prevent the perpetuation of poor maternal care across generations.”

The study authors demonstrated intergenerational epigenetic effects, and missed an opportunity to also investigate transgenerational epigenetically inherited effects. They cited reference 60 for the first part of the above quotation, but that reviewer misused the transgenerational term by applying it to grand-offspring instead of the great-grand-offspring.

There were resources available to replicate the study authors’ previous findings, which didn’t show anything new. Why not use such resources to uncover evidence even more applicable to humans by extending experiments to great-grand-offspring that have no potential germline exposure to the initial damaging cause?

Could a study design similar to A limited study of parental transmission of anxiety/stress-reactive traits have been integrated? That study’s thorough removal of parental behavior would be an outstanding methodology to confirm by falsifiability whether parental behavior is both an intergenerational and a transgenerational epigenetic inheritance mechanism.

Rodent great-grand-offspring can be studied in < 9 months. It takes > 50 years for human studies to reach the transgenerational generation. Why not attempt to “prevent the perpetuation of poor maternal care across generations?”

Isn’t it a plausible hypothesis that humans “with a history of maltreatment mistreat their own offspring?” Isn’t it worth the extra effort to extend animal research to investigate this unfortunate chain? “Pharmacological manipulation of DNA methylation normalizes maternal behavior, DNA methylation, and gene expression in dams with a history of maltreatment”