A cherry-picked DNA methylation study

This 2020 US/Sweden/Denmark human study measured twins during their old age:

“We evaluate individual differences in DNA methylation at individual CpG sites across the methylome across 10 years in two Scandinavian samples of same‐sex aging twins. We test two competing hypotheses about the longitudinal stability and change in DNA methylation:

  1. The contribution of genetic influences changes with age, reflecting diminishing influence across time; and
  2. Nonshared factors accumulate in importance, signaling an increasing diversity of response to environmental exposures.

Understanding epigenetic changes over time in the elderly may identify pathways of decline or plasticity (e.g., maintenance or even boosts in functioning) during the aging process and help with elucidating the biology of aging and survival.

Across time, stability in methylation is primarily due to genetic contributions, while novel experiences and exposures contribute to methylation differences. Elevated genetic contributions at age‐related methylation sites suggest that adaptions to aging and senescence may be differentially impacted by genetic background.”

https://onlinelibrary.wiley.com/doi/full/10.1111/acel.13197 “A decade of epigenetic change in aging twins: Genetic and environmental contributions to longitudinal DNA methylation”


Swedish subject measurements were taken at ages 62 and 72. Danish subject measurements were taken at ages 76 and 86.

One epigenetic clock that used 2019 technology was favored over three others, including Horvath’s 2013 original clock. For some reason this study didn’t use his 2018 skin-and-blood clock that had vastly improved technology such as an 18-fold increase in genomic coverage with Illumina 450k/850k bead arrays.

These researchers’ intentions became evident with:

“The 353 Horvath clock sites were selected as best predictors of chronological age using multiple tissues. The 71 Hannum clock sites best predicted age (adjusted for sex, BMI) based on methylation observed in whole blood while the 514 sites from the Zhang prediction model relied on methylation observed in blood and saliva samples (Zhang et al., 2019).

The current findings of moderately higher heritabilities in the Zhang and Hannum sites versus the other clock sites may be in part due to our use of blood tissue.”

The 18-fold increase improved accuracy in blood for the 2018 Horvath clock. Could these researchers ignore it and claim they did their due diligence in 2019 and 2020?


A larger issue was this study’s duality paradigm of either heritability or environment being solely responsible for observed changes. Consider what A blood plasma aging clock found at ages 60 and 78 peaks:

The above changes were due to life stage. Josh Mitteldorf did his usual excellent job of providing contexts for that study with New Aging Clock based on Proteins in the Blood, including:

“The implication is that a more accurate clock can be constructed if it incorporates different information at different life stages. None of the Horvath clocks have been derived based on different CpG sites at different ages, and this suggests an opportunity for a potential improvement in accuracy.”

Weren’t changes in subjects’ life stages relevant to their epigenetic changes? Why wouldn’t their life stages have been among the causes of observed effects?

Sulforaphane and RNAs

This 2020 Texas review subject was long non-coding RNAs:

“We review the emerging significance of long non-coding RNAs (lncRNA) as downstream targets and upstream regulators of the Nrf2 signaling pathway, a critical mediator of diverse cellular processes linked to increased cell survival.

It is believed that more than 3% of human genes are regulated by the Nrf2/Keap1 pathway. In addition to the classical cytoprotective and oxidative stress response genes transactivated by Nrf2, emerging evidence suggests a role for non-coding transcript regulation at the level of noncoding RNAs, [which] far outnumber protein-coding genes in the human genome.

One important distinction between miRNAs and lncRNAs is that the latter are often species-specific, meaning that a human lncRNA typically cannot be studied in the mouse or rat, and vice versa.

Sulforaphane (SFN) acts via multiple mechanisms to modulate gene expression, including the induction of Nrf2-dependent signaling. In addition to the established canonical targets of Nrf2, such as NQO1 and HMOX1, SFN altered the expression of multiple lncRNAs.

Given that SFN induces NMRAL2P [a lncRNA pseudogene] and several other lncRNAs in colon cancer cells, further studies are warranted on their respective roles as upstream regulators and/or downstream targets of Nrf2 signaling.

Pharmacological modulation of Nrf2 is considered a viable strategy against chronic conditions that are accompanied by oxidative stress and inflammation:

  • DMF [dimethyl fumurate] is the most successful Nrf2 activator, FDA-approved in 2013 for the treatment of relapsing remitting multiple sclerosis. However, DMF causes leukopenia and other side-effects.
  • Bardoxolone cleared Phase II clinical trials for the treatment of advanced chronic kidney disease and type 2 diabetes mellitus, but was halted in Phase III trials due to cardiovascular concerns.
  • SFN is relatively unstable at room temperature.

We used reported bioinformatics approaches to search for putative ARE [antioxidant response element] sequences among the entire set of 16,000+ annotated human lncRNAs. 13,285 promoter regions contained one or more potential binding sites for Nrf2.”

https://www.sciencedirect.com/science/article/pii/S0304383520303670 “Emerging crosstalk between long non-coding RNAs and Nrf2 signaling”


This study hyped lncRNAs in that only 7 have been validated as Nrf2 targets, and 8 validated as Nrf2 regulators. For regulators, “protein and/or miRNA interacting partners are yet to be fully corroborated” as well.

Also, there’s no need for a “SFN is relatively unstable at room temperature” problem. Just create sulforaphane right before consuming it.

Twice a day I microwave an average 65.5 grams of 3-day-old broccoli sprouts immersed in 100 ml water with a 1000W microwave on full power for 35 seconds to achieve 60°C. After microwaving I transfer broccoli sprouts to a strainer, and wait five minutes to allow further myrosinase hydrolization of glucoraphanin and other glucosinolates into sulforaphane and other healthy compounds.

Reprogram inflammation with β-glucan

This 2020 French human cell study found:

“Exposure of mononuclear phagocytes to β-glucan contributes to the induction of innate immune memory, which is associated with long-term epigenetic, metabolic, and functional reprogramming. We investigated how preincubation of human monocytes with particulate β-glucan affects the biological response of macrophages following NLRP3 inflammasome activation.

Upon infection or cellular damage, NLRP3 assembles into a multiprotein inflammasome complex leading to the release of IL-1β. However, NLRP3 inflammasome activity can also be detrimental to the host, and its aberrant chronic activation is associated with severe pathologies.

β-Glucan is a safe molecule present in food products and already widely used in food supplementation. Although β-glucan–induced innate memory is associated with a nonspecific protective effect against infections, the role of this functional reprogramming in autoinflammatory disorders is unknown.

Because of the administration frequency and conservation needs, IL-1β–targeted therapy is invasive, complex, and also costly. In addition, IL-1β, an acute-phase protein, is crucial for effective immune responses to infection, and inhibitors targeting IL-1β may lead to unintended immunosuppressive effects in addition to preventing NLRP3 inflammasome activity in itself.

Targeting the origin of the disease, i.e., NLRP3, would represent the best therapeutic strategy. Most of these candidate drugs directly interact with NLRP3, but none seems to regulate the early activation events upstream of NLRP3 inflammasome assembly.

β-Glucan acted upstream of the NLRP3 inflammasome. β-glucan–induced innate immune memory represses IL-1β–mediated inflammation and support its potential clinical use in NLRP3-driven diseases.”

https://www.jci.org/articles/view/134778 “β-Glucan–induced reprogramming of human macrophages inhibits NLRP3 inflammasome activation in cryopyrinopathies”


This study came closer to addressing causes than others with:

“Targeting the origin of the disease would represent the best therapeutic strategy.”

It’s apparently too recent with a July 27th published date to make it onto https://www.betaglucan.org/i-p/, but earlier β-glucan inflammation studies may be found there.

Topical sulforaphane protects skin

This 2020 Rutgers rodent study explored topical application of sulforaphane to prevent UVB-induced skin carcinogenesis:

“We investigated the transcriptomic and DNA methylomic changes during tumor initiation, promotion, and progression and its impact and reversal by sulforaphane (SFN). The production of ROS and inflammation are closely linked to UVB-induced carcinogenesis. SFN protects skin cells from UVB-induced damage mainly through promoting anti-inflammatory, antioxidative and anticancer pathways.

We observed the changes after 2, 15 and 25 weeks of UVB exposure, which would represent the three different stages of skin cancer development. After 2 weeks of UVB exposure, we did not observe any obvious tumors in the UVB group. But after 15 weeks of UVB exposure, some obvious tumors were observed in the skin.

After 15 weeks of UVB treatment in epidermal tissue, the difference between the UVB group and the control group was significantly more than that between the SFN group versus the UVB group. SFN appears to have better cancer-protective effects in earlier time points (weeks 14 and 20) than later time point (week 24). At weeks 20, SFN had significantly fewer tumors with decreased total tumor volume and tumor number.

SFN plays a highly regulatory role in various signaling pathways during UVB irradiation. SFN impacts UVB-induced alterations of DNA methylation profiles, and importantly, SFN treatment attenuates some of these DNA methylation changes. We found a subset of genes associated with SFN treatment, and the relevant changes in gene expression may be driven by promoter CpG methylation status.”

https://cancerpreventionresearch.aacrjournals.org/content/13/6/551 “Epigenome, Transcriptome, and Protection by Sulforaphane at Different Stages of UVB-Induced Skin Carcinogenesis” (not freely available)


We’re getting closer to using epigenetic clocks in sulforaphane studies. This study ignored the 2018 A multi-tissue full lifespan epigenetic clock for mice in favor of their homegrown DNA methylation measurements.

A search of ClinicalTrials.gov didn’t turn up directly relevant human studies.

Supporting individuals

This past Saturday evening into night I walked five miles over three hours in Manchester, New Hampshire, with two individuals. Several items of interest, incidental to our enjoyable experiences:

  • My first impression was that it could have been this time last year. People who had spent a long winter and spring indoors were happy to be outdoors.
  • There were small differences from 2019 in that outdoor and indoor tables were widely spaced, and a few customers wore masks. We ate a meal indoors that included good paella, ceviche, and calamari.
  • I didn’t see instances of violence or property crimes.
  • I observed social deviancy in ~1% of the people. A crazy person talked to herself while walking down the sidewalk carrying a plastic garbage bag full of who-knows-what – worldly belongings? One homeless person slept off a binge, another engaged in a binge with a bottle in front of him, while a third walked glassy-eyed, accompanied by counsel.

I didn’t see any people my chronological age outdoors. Okay, it was Saturday night.


I encourage every individual to take responsibility for every aspect of each of our own one precious life.

I strongly object to destroying society among individuals, especially young people’s social and economic lives. Current cover stories promise to destroy a person’s social presence, reputation, residence, economic development – whatever it takes – to conform them to the desired “norm.”

Should we destroy society to ostensibly (“outwardly appearing as such; professed; pretended“) protect people who don’t take responsibility for themselves?

  • For our own non-communicable diseases like Type 2 diabetes, obesity, etc., that make us susceptible to other diseases?
  • For our own neglected health issues that we look to others to resolve instead of looking to ourselves?

Will Part II be governments granting themselves even more powers with a cover story that they will restore the order they destroyed?


The individuals I walked with support open-carry firearms. Instances of property crimes and violence I saw: 0 among ~400 people outdoors. I saw three that supported open carry:

  1. A pub owner or manager;
  2. A person on Manchester’s main street; and
  3. A person shouting from their car.

Instances of property crimes and violence in US cities on Saturday night who prohibit open carry? Look up:

  • Seattle (at least 59 people injured by mob violence);
  • Portland (another day after day, week after week, month after month of unarrested mob property crimes and violence);
  • Chicago (another day that added to the 2,200+ people shot by gangs during 2020).

What went on in your city this past Saturday night?

Politically correct about erucic acid and broccoli seeds

To follow up Caution on broccoli seed erucic acid content? this 2020 German review sympathetically analyzed government overreach on erucic acid contents in several foods:

“We measured exemplarily samples of rapeseed, mustard, further Brassicaceae and used the data to discuss possible consequences for consumers, producers and the food sector. This data was supplemented with possible analytical problems.

The new and lower erucic acid level in the EU is anticipated but will increase the need of an efficient control system by producers and food processors in order to avoid violations of erucic acid limit values and sale bans. The new proposed legislation will likely prompt some producers to reformulate their recipes, which can be achieved by lowering the fat content or by moving to mustard seeds with lower erucic acid content.

The amount of erucic acid in fish should not be neglected.”

https://www.sciencedirect.com/science/article/pii/S235236462030002X “Erucic acid in Brassicaceae and salmon – An evaluation of the new proposed limits of erucic acid in food” (not freely available)


The paper didn’t measure erucic acid percentages of total fatty acids in broccoli seeds. Also noticeably absent were analyses of animal studies performed a long time ago that formed the bases of current government actions.

Nothing to see here, move along. Much more effort was put into creating new health hazards for consumers, as if we should now be required to worry about eating salmon.

The 2017 position paper establishing erucic acid limits was excessively cited twelve times, such as for:

“Likewise, broccoli seeds were high in erucic acid but this fatty acid was not detected in edible parts of the vegetable. Hence, intake of erucic acid via these vegetables seems to be irrelevant.”

I mentioned problems in the poorly-evidenced 2002 study cited by this position paper. That researcher couldn’t be bothered to use just one broccoli cultivar for only three measurements, or disclose broccoli sprout age. But apparently it’s a fait accompli, elevated to an indisputable fact.

Despite many technical details, the current paper was politics. It detracted from science, with a cover story “in favor of consumer protection.” These researchers descended further into advocacy with “analysis” beginning with:

“Imagine (cruciferous) vegetables having an erucic acid content of 50% in the lipids.”

They did cause me to “imagine” eating hot dogs with mustard. But maybe that’s because baseball season is finally starting?


These vendors of broccoli seed powder don’t seem concerned about disclosing erucic acid content. What do you think?

🙂

 

Caution on broccoli seed erucic acid content?

1. While looking through PubMed “broccoli skin” search results, I read a 2018 study Comparative Study of Predominant Phytochemical Compounds and Proapoptotic Potential of Broccoli Sprouts and Florets that cautioned about erucic acid content in broccoli seeds:

“Our results revealed significantly higher total UFAs [unsaturated fatty acids] content in the sprouts in comparison to the florets, with very low amounts of harmful erucic [27] acid in sprouts (0.5%) and florets (2%), in comparison to the broccoli seeds (38% – data not shown).”

But its cited reference [27] Various concentrations of erucic acid in mustard oil and mustard said nothing about broccoli seeds.

Values were on a dry weight basis. Broccoli sprout age was four days.

2. Another search found this 2017 Erucic acid in feed and food position paper which stated:

“When in this Scientific Opinion the erucic acid content is reported as a percentage, this value refers to the percentage erucic acid in the total fatty acids on a weight basis.

A tolerable daily intake of 7 mg/kg body weight per day for erucic acid was established.”

3. It referenced a 2002 Determination and Health Implication of the Erucic Acid Content of Broccoli Florets, Sprouts, and Seeds which stated:

“The erucic acid content of broccoli florets, sprouts, and seeds was found to be about 0.8, 320, and 12100 mg/100 g, respectively.”

Respective erucic acid percentages of total lipids on a fresh weight basis were provided as 0.4%, 1.1%, and 26.9%.

Florets, sprouts, and seeds had no relationships among them as they were different broccoli cultivars. Broccoli sprouts’ age wasn’t disclosed.

4. The 2002 study was updated in a 2004 Glucoraphanin and 4-Hydroxyglucobrassicin Contents in Seeds of 59 Cultivars of Broccoli, Raab, Kohlrabi, Radish, Cauliflower, Brussels Sprouts, Kale, and Cabbage which stated:

“All seed accessions contained substantial amounts of hexane-extractable lipids ranging from 21.8 to 42.0% (mean of 32.8%; 21.8-37.0 and 30.9% range and mean, respectively, for broccoli cultivars only), which were composed of 27.0-56.7% (mean of 46.7%;39.4-56.7 and 50.2% range and mean, respectively, for broccoli cultivars only) erucic acid.”

Seeds of the 2002 broccoli sprouts commercial product were measured at 31.4% lipids, with erucic acid content 51.6% of total lipids.

5. The 2018 study cited a 2013 Biochemical composition of broccoli seeds and sprouts at different stages of seedling development whose broccoli seed and sprout composition dry weights are in the below graphic:

  • Broccoli seed lipid percentage of total carbohydrates plus crude fiber would be 9.36 g / (58.89 g + 15.47 g) = 12.6%.
  • 3-day-old broccoli sprouts lipid percentage of total carbohydrates plus crude fiber would be 8.67 g / (54.4 g + 8.97 g) = 13.7%.
  • No erucic acid contents were disclosed.


These four studies all required further work:

  • 2002 couldn’t be bothered to use just one broccoli cultivar for its three measurements, or disclose broccoli sprout age.
  • 2004 couldn’t resolve many of their findings with other studies.
  • 2013 used weights to equate measurements, instead of relating germination stages back to a beginning number of seeds and their measurements.
  • 2018 provided a bogus reference and an unsupported “broccoli seeds (38% – data not shown).” It claimed similarity with 2013, but a statistics package would say otherwise. It also didn’t comply with disclosing fatty acids weight as a percentage of broccoli sprouts weight.

Home sprouting has to deal with:

  • unknown cultivar,
  • unknown glucoraphanin and other glucosinolates contents,
  • unknown sulforaphane and other healthy compounds, and now
  • unknown erucic acid content.

Let’s reverse Microwave broccoli seeds to create sulforaphane calculations with 3-day-old broccoli sprouts have the optimal yields information to estimate an erucic acid content in one tablespoon of broccoli seeds. Measurements from Week 18 and Week 19 of Changing to a youthful phenotype with broccoli sprouts.

  • Broccoli seed weight of one tablespoon 10.7 g.
  • Lipids weight (10.7 g x 12.6% [2013 study]) = 1.35 g.
  • Erucic acid weight in one tablespoon of broccoli seeds (1.35 g x 26.9% [2002 study]) = 0.36 g.

This 0.36 g erucic acid content would be lower than 2017 guidelines for my 70 kg weight (7 mg x 70) = 0.49 g.

Let’s reverse Estimating daily consumption of broccoli sprout compounds techniques to estimate an erucic acid content in my daily consumption of 3-day-old broccoli sprouts grown from two tablespoons of seeds:

  • 131 g 3-day-old broccoli sprouts.
  • Maximum lipids weight (131 g x 13.7% [2013 study]) = 17.9 g.
  • Maximum erucic acid weight in 3-day-old broccoli sprouts (17.9 g x 1.1% [2002 study]) = 0.20 g.

Plug in your own numbers, but it looks like caution isn’t warranted for broccoli seed consumption. Consequences of a possible erucic acid content may be less than broccoli seeds’ healthy aspects.

One mitigation may be to start germination. Pick a point between broccoli seeds’ % of total fatty acids and ending 0.5% of 4-day-old sprouts [2018 study].

Not concerned with a daily estimate < .49 g erucic acid for broccoli seeds and sprouts. Back to a PubMed “broccoli skin” search.

See Politically correct about erucic acid and broccoli seeds for a follow up.