Does reprogramming signaling pathways create memories?

This 2020 study investigated genes and signaling pathways for inflammatory memory:

“Fibroblast-like synoviocytes (FLS) play a critical role in pathogenesis of rheumatoid arthritis (RA). Chronic inflammation induces transcriptomic and epigenetic modifications that imparts a persistent catabolic phenotype to the FLS, despite their dissociation from the inflammatory environment.

Sustained activated genes established pro-inflammatory signaling components known to act at multiple levels of NF-κB, STAT and AP-1 signaling cascades. Sustained repressed genes included critical mediators and targets of the BMP [bone morphogenic protein] signaling pathway.

We identified sustained repression of BMP signaling as a unique constituent of the long-term inflammatory memory induced by chronic inflammation.

FLS are synovial tissue-resident and specialized mesenchymal cells critical for homeostasis. Key features of these cells during homeostasis include the production of extracellular matrix components and providing nutrients to the synovial fluid. Healthy synovium is composed of multiple layers of FLS, which forms the synovial lining and sublining through cell–cell contacts.

Inflammatory and pro-resolving mediators are tightly regulated to maintain normal synovium functioning. However, in inflammatory and autoimmune diseases such as rheumatoid arthritis, an imbalance between these signals causes homeostasis disruption leading to synovial tissue damage, cartilage destruction and bone degeneration.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679373/ “Chronic exposure to TNF reprograms cell signaling pathways in fibroblast-like synoviocytes by establishing long-term inflammatory memory”


These researchers described a positive feedback loop that kept rheumatoid arthritis in place. No feedback diagram or explanation of what sustained a disease condition other than to say:

“Gene expression changes induced by short-term tumor necrosis factor-alpha (TNF-α) treatment were largely sustained in the FLS exposed to chronic inflammation.”

Okay – then what upstream signals sustained TNF-α? What would it take to interrupt that feedback loop? What initiated it?

Studies usually substantiate effects by also developing evidence for causes of opposite effects and of no effects. This study investigated neither reversibility nor no effect, and instead stated:

“Multiple signaling networks are irreversibly modified due to TNF-α-mediated long-term epigenetic and transcriptomic reprogramming. We speculate that sustained repression of BMP signaling may be critically required to ensure the persistently transformed phenotype of RA FLS.”

No evidence was offered for “irreversibly modified.” Anyway, that didn’t fit with:

“We postulate that simultaneous targeting of these activated and repressed signaling pathways may be necessary to combat RA persistence.”

Enduring epigenetic memories? Or continuous toxic stimulation? provided another perspective: “Enduring epigenetic effects may be symptoms rather than causes when toxic conditions persist.”


Been on a Steely Dan kick lately. Probably due to this year’s Royal Scam:

Zinc and broccoli sprouts – a winning combination

This 2019 study deserved better coverage than a one sentence mention in Reversal of aging and immunosenescent trends with sulforaphane:

“Obstructive sleep apnea syndrome is one of the most common breathing disorders in sleep, with a high prevalence of 3–7% and severe consequences. It is characterized by intermittent hypoxia (IH) due to recurrent episodes of partial or complete collapse of the upper airway during sleep, leading to blood hypoxemia, hypercapnia, sleep fragmentation, augmented respiratory efforts, and increased sympathetic activity.

Our study is the first investigation of the combination of BSE [broccoli sprout extract] and Zn [zinc], Nrf2, and MT [metallothionein] inducers, to protect against IH-induced cardiomyopathy. By effectively activating Nrf2, its downstream targets, and MT, this combination can ameliorate defects associated with IH-induced cardiomyopathy more effectively than monotherapies.

Mice were administered with BSE (equivalent to SFN [sulforaphane] 2 mg/kg) and/or Zn sulfate heptahydrate (5 mg/kg) by gavage from 8 weeks of age at a frequency of once every other day for 8 weeks. Doses used in this study are safe to convert to human doses. [2 mg x .081 x 70 kg = 11 mg sulforaphane; 5 mg x .081 x 70 kg = 28 mg zinc]

  • Heart mass was significantly lower in the IH-BSE/Zn group than in IH and IH-BSE groups. Heart mass / tibia length ratio was significantly lower in the IH-BSE/Zn group than in IH and monotherapy groups.
  • Treatment with BSE and/or Zn can ameliorate myocardial fibrosis associated with IH, to a certain extent, and combination therapy has the best antifibrotic effect among treatments.
  • BSE or Zn can significantly ameliorate myocardial inflammation induced by IH, but the combination provides a better anti-inflammatory effect.
  • We used 3-NT as an indicator of the severity of oxidative stress. 3-NT protein levels were significantly reduced in IH mice for all treatment groups, and reduction was greater in the combination treatment group.
  • Combination is more effective than monotherapies to activate Nrf2-mediated antioxidant function.

  • In Zn-treated and combination treatment groups, MT protein expression was significantly higher than in the IH group, and there was only a slight increase in the IH-BSE group.”

Combination of Broccoli Sprout Extract and Zinc Provides Better Protection Against Intermittent Hypoxia-Induced Cardiomyopathy Than Monotherapy in Mice


One way to improve broccoli sprout compounds’ effects is to eat them with zinc. One way to improve zinc’s actions is to take it with broccoli sprouts.

Part 2 of Eat broccoli sprouts for your eyes

I was a little bothered by an unreferenced statement in Eat broccoli sprouts for your eyes that:

“Once AGEs are formed, most are irreversible.”

I searched curated 2020 studies for “revers” and found that recent blog studies favored reversibility of epigenetic changes 12-to-2. Do they reflect my selection bias, or is there something different about AGEs?

Let’s start with this statement:

“Although AGEs are irreversible adducts and cross-links in our tissues, these can be removed through different proteolytic capacities:

  • The ubiquitin proteasome system (UPS) – Ubiquitin is a protein that when conjugated to a protein substrate can facilitate degradation of that substrate by the proteasome. Obsolete or damaged proteins are tagged with ubiquitin and these ubiquitinated substrates are degraded by the proteasome. Operates mainly on soluble substrates.
  • Autophagy – Can operate on insoluble substrates, including organelles such as mitochondria. Autophagy requires macromolecular assemblies and organelles to identify, sequester, and eventually degrade substrates via the lysosome.

Unfortunately, the function of both proteolytic pathways declines with extensive glycative stress and upon aging in many tissues, resulting in intracellular accumulation of protein aggregates (also glycated conjugates) and dysfunctional organelles. This thwarts strategies to lower AGEs accumulation by boosting proteolytic capacities.”

https://www.mdpi.com/2076-3921/9/11/1062/htm “Glyoxalase System as a Therapeutic Target against Diabetic Retinopathy”


So humans can remove irreversible AGE epigenetic changes as long as the individual isn’t too stressed or old? Studies from 2008 to 2012 were cited for the above statement and graphic.

Citation 211 Sulforaphane delays diabetes-induced retinal photoreceptor cell degeneration (not freely available) 2020 findings were instructive:

“SF [sulforaphane] can delay photoreceptor degeneration in diabetes. The underlying mechanism is related to:

  • Inhibition of ER [endoplasmic reticulum] stress;
  • Inflammation; and
  • Txnip [thioredoxin-interacting protein] expression through activation of the AMPK [adenosine 5′-monophosphate (AMP)-activated protein kinase] pathway.

Function of the retina in diabetic [DM] mice as determined by ERG [electroretinography].”


This chart demonstrated that preventing diabetes’ retinal effects (non-diabetic control bar) was measurably better than trying to fix them. Are future choices of humans who give themselves this non-communicable disease also limited to addressing symptoms?

The AMPK pathway was mentioned in:

  1. Reversal of aging and immunosenescent trends with sulforaphane:”

    Dihydroxyvitamin D3 and sulforaphane are compounds that safely induce AMPK activation, and may have wide-ranging implications for both normal and pathological aging.”

  2. Part 2 of Reversal of aging and immunosenescent trends with sulforaphane:

    “NQO1 plays a key role in AMPK-induced cancer cell death through the CD38/cADPR/RyR/Ca2+/CaMKII signaling pathway. Expression of NQO1 is elevated by hypoxia / reoxygenation or inflammatory stresses through nuclear accumulation of the NQO1 transcription factor, Nrf2. Activation of the cytoprotective Nrf2 antioxidant pathway by sulforaphane protects immature neurons and astrocytes from death caused by exposure to combined hypoxia and glucose deprivation.”

This first example was vitamin D3’s separate yet connected signaling pathway that acts both additively and synergistically with broccoli sprout compound effects. Followed by signaling pathways becoming cascadingly activated from sulforaphane’s main effect, Nrf2 signaling pathway activation.


Mild stress improves broccoli compound yields

This 2020 plant study by the same university as Our model clinical trial for Changing to a youthful phenotype with broccoli sprouts investigated seasonal and stressful effects on broccoli compounds:

“In this study, three crop trials were carried out to evaluate effects of cultivation season, application of different dosages of methyl-jasmonate (MeJA) on overall quality and on total content of bioactive compounds of ‘Parthenon’ broccoli cultivated under field conditions of southeastern Spain.

Elicitation is the main tool used to increase content of secondary metabolites in vegetables, as it induces stress responses in plants. Several studies have involved application of elicitors to broccoli plants in order to improve their nutritional properties (although this application is more common for seeds and sprouts).

Content of total carotenoids, phenolic compounds and glucosinolates were higher in autumn compared with spring, showing increases of 2.8-fold, 2-fold and 1.2-fold, respectively. Moreover, a double application of MeJA increased contents of total carotenoids, phenolic compounds and glucosinolates by 22%, 32% and 39%, respectively, relative to untreated samples.

Controlled and timely (four days before harvest) application of 250 µM MeJA as an elicitor to aerial parts of plants, on two consecutive days, yielded florets of Parthenon broccoli with higher contents of bioactive compounds, without changing its overall quality.”

https://www.mdpi.com/2304-8158/9/10/1371/htm “Seasonal Variation of Health-Promoting Bioactives in Broccoli and Methyl-Jasmonate Pre-Harvest Treatments to Enhance Their Contents”


Findings by broccoli compound category were:

Glucosinolates

“Total content of GLSs was 2-fold higher in autumn than in spring. Total precipitation [2018] in spring was 361 mm compared with 185 mm in autumn. The water deficit in autumn could have contributed to the increase in total GLS content.

The main compound in samples of plants cultivated in spring (first and third assays) was glucoiberin (GIB), followed by glucoraphanin (GRA). The order was reversed in broccoli cultivated in autumn, with GRA being the main compound, followed by GIB, for all treatments.

Aliphatic GLSs were predominant in our Parthenon samples, representing on average 76%, 86% and 83%, of total GLSs in the first, second and third assays, respectively. In relation to the effect of MeJA on content of GLSs, neoglucobrassicin (NGB) was the only compound that showed a significant increase after application of MeJA in seasonal trials, since other GLSs decreased or did not differ with respect to the control group.

NGB increased significantly, from 0.3 mg/kg f.w., to 175 mg/kg f.w. in broccoli treated with two consecutive doses of 250 μM MeJA, and contents of GBSs, total indole GLSs and total GLSs also increased. In contrast, one single dose of 500 μM MeJA did not enhance contents of these compounds.”

Phenolics

“Contents of flavonols and chlorogenic acids were higher in autumn than in spring, whereas content of sinapic acid derivatives was higher in spring. Influence of light on individual phenolic compounds could explain the increase in flavonols and chlorogenic acid derivatives in autumn.

Although MeJA altered contents of phenolic compounds, this effect was not clearly associated solely with MeJA. We found a greater effect of the excipient and MeJA in autumn. When we added an extra stress factor – namely, MeJA – the impact was not as great as in autumn.”

Carotenoids

“In broccoli cultivated in spring, the order was β-carotene > lutein > violaxanthin > neoxanthin, while in autumn the order was β-carotene > violaxanthin > lutein > neoxanthin. Content of total carotenoids in broccoli cultivated in autumn (26 mg/kg) represented a 2.8-fold increase compared to broccoli grown in spring (9 mg/kg).

Treatment with MeJA significantly reduced total content of carotenoids in broccoli cultivated in autumn, whereas it did not show any effect on plants cultivated in spring, and in some cases even led to an increase in carotenoid content. Plants that received two applications of 250 µM MeJA content of carotenoids (34 mg/kg f.w.) increased in comparison with plants without this treatment (28 mg/kg f.w.) as well as those receiving one application of 500 µM MeJA (28 mg/kg f.w.).

Chlorophyll content was directly related to carotenoids content, with a strong correlation in autumn. Carotenoids absorb solar light in the spectral region not covered by chlorophylls and pass light energy to chlorophyll a, protecting it from harmful reactions that occur in conditions of excessive light, in the presence of oxygen. When high temperatures reduce content of carotenoids in spring, a reduction in total chlorophylls is also observed, possibly due to the photo-oxidation process.”


Sulforaphane in the Goldilocks zone

This 2020 paper reviewed hormetic effects of a broccoli sprout compound:

“Sulforaphane (SFN) induces a broad spectrum of chemoprotective effects across multiple organs that are of importance to public health and clinical medicine. This chemoprotection is dominated by hormetic dose responses that are mediated by the Nrf2/ARE pathway and its complex regulatory interactions with other factors and pathways, such as p53 and NF-κB.

The stimulatory zone for in vitro studies proved to be consistently in the 1-10 μM range. Hormetic studies of SFN strongly targeted activation of Nrf2.

Capacity to activate Nrf2 diminishes with age, and may affect capacity of SFN to effectively enhance adaptive responses.

A 4-hour exposure induced a 24 hour Nrf2-mediated increase in enzymes that reduce free-radical damage in neurons and astrocytes. Repeated 4-hour treatment for four days affected an accumulation along with a persistent protection.

In the case of continuous exposure to SFN, such as taking a daily supplement, SFN treatment did not result in an accumulation of HMOX1 [heme oxygenase (decycling) 1 gene] mRNA or protein. This suggested that HMOX1 response may experience feedback regulation, avoiding possible harmful overproduction.”

https://www.sciencedirect.com/science/article/abs/pii/S1043661820315917 “The phytoprotective agent sulforaphane prevents inflammatory degenerative diseases and age-related pathologies via Nrf2-mediated hormesis” (not freely available)


One coauthor has been on a crusade to persuade everybody of this paradigm. Hormesis’ hypothesis isn’t falsifiable in all circumstances, however.

Hormetic effects may be experimental considerations. But what’s the point of performing sulforaphane dose-response experiments in contexts that are physiologically unachievable with humans? Two examples:

  1. Autism biomarkers and sulforaphane:

    “There was no concentration-dependence in the induction of any of the genes examined, with the higher (5 μM) concentration of SF even showing a slightly diminished effect for the induction of AKR1C1 and NQO1. Although this concentration is achievable in vivo, more typical peak concentrations of SF (and its metabolites) in human plasma are 1-2 μM.”

  2. Human relevance of rodent sulforaphane studies:

    “Over two-thirds of the animal studies have used doses that exceed the highest (and bordering on intolerable) doses of sulforaphane used in humans. The greater than 4-log spread of doses used in mice appears to be driven by needs for effect reporting in publications rather than optimization of translational science.”

This paper cited many hormetic effects that were human-irrelevant without making a distinction. It also had parts such as:

“The capacity for high concentrations of AITC [allyl isothiocyanate] to enhance genetic damage is not relevant since such high concentrations are not realistically achievable in normal human activities.

Humans ingest only the R-isomer of SFN via diet. Their dosing strategy adopted concentrations of R-SFN that were less than those employed to induce cytotoxic effects in cancer cells and that simulated its consumption as a dietary supplement.”


Landing eagle

DIM effects on BRCA carriers

This 2020 study evaluated a broccoli sprout compound’s effects on breast cancer development:

“Women who carry the BRCA mutation are at high lifetime risk of breast cancer, but there is no consensus regarding an effective and safe chemoprevention strategy. A large body of evidence suggests that 3,3-diindolylmethane (DIM), a dimer of indole-3-carbinol found in cruciferous vegetables, can potentially prevent carcinogenesis and tumor development.

A year’s supplementation with DIM 100 mg daily in BRCA carriers was associated with a significant decline in FGT [fibroglandular tissue] amount on MRI. Larger randomized studies are warranted to corroborate these findings.”

https://academic.oup.com/carcin/article/41/10/1395/5847633 “3,3-Diindolylmethane (DIM): a nutritional intervention and its impact on breast density in healthy BRCA carriers. A prospective clinical trial”


This study didn’t address DIM bioavailability. What were the DIM amounts each subject actually processed? How was DIM bioavailability related to their “significant decline in FGT” outcome?

Studies that found DIM was only 1-3% bioavailable after oral administration include:

PubChem lists DIM molecular weight as 246.31 g/mol. A 4.06 μmol DIM amount (.001 / 246.31) equals a 1 mg weight. The study’s daily DIM intake 100 mg weight was a 406 μmol amount.

Eat broccoli sprouts for DIM and Our model clinical trial for Changing to a youthful phenotype with broccoli sprouts measured DIM excreted as a result of eating 30 grams raw broccoli super sprouts every day. Indolic glucosinolate precursors of DIM were as follows:

indolic glucosinolates

DIM at the 70-day point was an average 0.650 μmol amount, which was almost twice those subjects’ 0.334 average beginning amount. If each subject’s DIM was collected over 24-hours, using precursor conversion calculations may have produced bioavailability measurements.


Young dolphins eating breakfast

Eat broccoli sprouts for pain?

This 2018 study investigated pain-relieving effects of two broccoli sprout compounds, sulforaphane and chlorogenic acid:

“Pharmacological evidence of the antinociceptive properties of broccoli aqueous extracts and bioactive metabolites were investigated in an experimental model of pain.

It was found that sprouts produced better antinociceptive response than seeds and inflorescence of broccoli, where SFN [sulforaphane] and CA [chlorogenic acid] were partial responsible. Opioid receptors were implicated in the antinociceptive effect of SFN, whereas calcium channels were involved in the concentration-dependent spasmolytic activity.

Our results give evidence of a dose-dependent antinociceptive effect of CA that might act in a synergic interaction with SFN and other metabolites to produce antinociceptive activity.”

https://www.sciencedirect.com/science/article/abs/pii/S0753332218333286 “Broccoli sprouts produce abdominal antinociception but not spasmolytic effects like its bioactive metabolite sulforaphane” (not freely available)


8-day-old broccoli sprouts were treated Days 5-8 with methyl jasmonate to increase glucosinolates as Our model clinical trial for Changing to a youthful phenotype with broccoli sprouts did.

I hadn’t previously noticed papers on “Chlorogenic and Sinapic acid derivatives” that are part of my daily intake, but there’s much recent research. Consider these October 2020 chlorogenic acid papers for example:


I found If it stinks, it’s good for you as a result of it citing this study. See Broccoli sprout compounds include sinapic acid derivatives to follow on that subject.

I rated this study as Required further work. This is my 31st week of eating a clinically relevant amount of broccoli sprouts every day, and I still take acetaminophen.

Anti-tumor effects of β-glucan

This comprehensive 2020 rodent study investigated dozens of scenarios for β-glucan in the context of anti-tumor immunity:

“Neutrophils and granulopoietic progenitors are major cellular effectors of β-glucan-induced trained immunity. The anti-tumor effect of β-glucan-induced trained immunity was mediated by qualitative changes in neutrophils.

A tumor-suppressive phenotype in neutrophils was associated with training of granulopoiesis mediated by type I IFN [interferon] signaling. Our analysis provided additional evidence for trained immunity-induced epigenetic rewiring of granulopoiesis toward an anti-tumor phenotype and corroborated the experimentally demonstrated IFN- and ROS-related mechanisms.

We observed inhibition of tumor growth by systemic transfer of trained neutrophils into already tumor-bearing mice. As granulocyte transfusion is currently considered as a therapy in humans with neutropenia, it is conceivable that cancer patients could receive as an adjuvant immunotherapy granulocytes from normal donors after induction of trained immunity in the latter.

Our study is the first to link the anti-tumor actions of β-glucan to trained immunity. We show here that the innate immune training and rewiring of granulopoiesis underlies the anti-tumor effect of β-glucan.”

https://www.cell.com/cell/fulltext/S0092-8674(20)31299-X “Innate Immune Training of Granulopoiesis Promotes Anti-tumor Activity”


Which do you prefer? The study’s graphical abstract:

or one of its volcano plots?

Here’s an overview of one investigated direction:

“To determine whether adaptive immunity is involved in the anti-tumor effect induced by β-glucan, mice that lack B and T cells were treated with β-glucan [1 mg] prior to the secondary tumor challenge. Pre-treatment with β-glucan decreased both B16-F10 [melanoma] and LLC [Lewis lung carcinoma] tumor burden also in [these] mice, showing that the anti-tumor effect of β-glucan-induced trained immunity does not require adaptive immunity.”


This study provided another example of what they called rewiring (but I term reprogramming) of the body’s environmental signaling pathways to achieve a desired phenotype, trained innate immunity. Whatever the terminology, almost every day over the past fifteen years I’ve eaten β-glucan in an oats breakfast and a 1/3, 1/6 yeast supplement at dinner as part of individually evolving.

Reprogramming other signaling pathways are in blog posts such as:

Take responsibility for your one precious life.

Flying over waves

Dietary contexts matter

Two papers illustrated how actions of food compounds are affected by their contexts. The first was a 2020 UCLA rodent study:

“Long-chain polyunsaturated fatty acids (PUFAs), particularly omega-3 (n-3) PUFAs, have been indicated to play important roles in various aspects of human health. Controversies are observed in epidemiological and experimental studies regarding the benefits or lack of benefits of n-3 PUFAs.

Dietary docosahexaenoic acid (DHA; 22:6 n-3) supplementation improved select metabolic traits and brain function, and induced transcriptomic and epigenetic alterations in hypothalamic and hippocampal tissues in both context-independent and context-specific manners:

  • In terms of serum triglyceride, glycemic phenotypes, insulin resistance index, and memory retention, DHA did not affect these phenotypes significantly when examined on the chow diet background, but significantly improved these phenotypes in fructose-treated animals.
  • Genes and pathways related with tissue structure were affected by DHA regardless of the dietary context, although the direction of changes are not necessarily the same between contexts. These pathways may represent the core functions of DHA in maintaining cell membrane function and cell signaling.
  • DHA affected the mTOR signaling pathway in hippocampus. In the hypothalamus, altered pathways were more related to innate immunity, such as cytokine-cytokine receptors, NF-κB signaling pathway, and Toll-like receptor signaling pathway.

DHA exhibits differential influence on epigenetic loci, genes, pathways, and metabolic and cognitive phenotypes under different dietary contexts.”

https://onlinelibrary.wiley.com/doi/10.1002/mnfr.202000788 “Multi‐tissue Multi‐omics Nutrigenomics Indicates Context‐specific Effects of DHA on Rat Brain” (not freely available)


A human equivalent age period of the subjects was 12 to 20 years old. If these researchers want to make their study outstanding, they’ll contact their UCLA colleague Dr. Steven Horvath, and apply his new human-rat relative biological age epigenetic clock per A rejuvenation therapy and sulforaphane.

The second paper was a 2016 review Interactions between phytochemicals from fruits and vegetables: Effects on bioactivities and bioavailability (not freely available):

“The biological activities of food phytochemicals depend upon their bioaccessibility and bioavailability which can be affected by the presence of other food components including other bioactive constituents. For instance, α-tocopherol mixed with a flavonol (kaempferol or myricetin) is more effective in inhibiting lipid oxidation induced by free radicals than each component alone.

Interactions of phytochemicals may enhance or reduce the bioavailability of a given compound, depending on the facilitation/competition for cellular uptake and transportation. For example, β-carotene increases the bioavailability of lycopene in human plasma, and quercetin-3-glucoside reduces the absorption of anthocyanins.

Combinations of food extracts containing hydrophilic antioxidants and lipophilic antioxidants showed very high synergistic effects on free radical scavenging activities. A number of phytochemical mixtures and food combinations provide synergistic effects on inhibiting inflammation.

More research should be conducted to understand mechanisms of bioavailability interference considering physiological concentrations, food matrices, and food processing.”


Each of us can set appropriate contexts for our food consumption. Broccoli sprout synergies covered how I take supplements and broccoli sprouts together an hour or two before meals to keep meal contents from lowering sulforaphane bioavailability.

Combinations of my 19 supplements and broccoli sprouts are too many (616,645) for complete analyses. Just pairwise comparisons like the second paper’s example below would be 190 combinations.

binary isobologram

Contexts for each combination’s synergistic, antagonistic, or additive activities may also be influenced by other combinations’ results.

My consumption of flax oil (alpha linolenic acid C18:3) probably has effects similar to DHA since it’s an omega-3 PUFA and I take it with food. The first study’s human equivalent DHA dose was 100mg/kg, with its citation for clinical trials stating “1–9 g/day (0.45–4% of calories) n-3 PUFA.”

A 2020 review Functional Ingredients From Brassicaceae Species: Overview and Perspectives had perspectives such as:

“In many circumstances, the isolated bioactive is not as bioavailable or metabolically active as in the natural food matrix.”

It discussed categories but not combinations of phenolics, carotenoids, phytoalexins, terpenes, phytosteroids, and tocopherols, along with more well-known broccoli compounds.


Diving for breakfast

Rub some broccoli sprouts on it

This 2020 human/rodent study investigated treating and preventing skin photodamage with sulforaphane:

“Alterations in NRF2 signaling have been implicated in aging and stress-induced skin pigmentation disorders in the skin and hair follicles. NRF2 signaling regulates transcriptional programs involved in adaption and survival of cells in the setting of oxidative stress, and oxidative stress occurs in the setting of photodamage.

[1st human experiment with 14 subjects] Expression levels of NRF2 and its target heme-oxygenase-1 (HO-1) were evaluated by immunofluorescence (IF) in skin biopsies. Expression of NRF2 and HO-1 was significantly reduced in skin from individuals >45 years old.

[2nd human experiment with 7 different subjects] The left arm was chosen for treatment with BSE [broccoli sprout extract], as there is typically more photodamage on the left arm due to chronic sun exposure through the car window while driving in the US. A photoprotected area of skin on an upper inner arm was also treated.

Expression of total NRF2 and phosphorylated NRF2 (NRF2-P) by IF microscopy was detected at low baseline levels in photoprotected skin, suggesting some activity of the pathway, whereas the expression of total NRF2 and NRF2-P was undetectable in untreated photoexposed skin (Un). There was significantly elevated IF expression and fold change of IF signal of NRF2 and especially NRF2-P in SF [sulforaphane]-treated skin compared with Un skin in most individuals.

There was no evidence of increased total NRF2 or NRF2-P expression in SF-treated photoexposed skin in 2 individuals. There was also no significant improvement in mottled hyperpigmentation or difference in melanin deposition following SF treatment.

[Six mouse confirmation/exploratory studies] SF is known to have several non-NRF2–mediated targets, such as NF-κB and AP-1. However, our findings suggest that negative regulation of UV-mediated hyperpigmentation observed following SF treatment is occurring in an NRF2-dependent fashion:

  • UVB+SF treatment resulted in more than a 50% decrease in skin pigmentation and melanin deposition, indicating that SF could prevent UVB-induced skin pigmentation.
  • The therapeutic effect of SF on reducing UVB-induced skin pigmentation was dependent on keratinocyte-intrinsic IL-6 receptor α (IL-6Rα) signaling that upregulated NRF2, which led to inhibition of melanogenesis.

Our results provide direct in vivo evidence of how NRF2 is involved in response to oxidative stress associated with photodamage and chronic UV exposure. Treatment of human or mouse skin hyperpigmentation with SF provided the proof of concept for targeting the NRF2 pathway as a therapeutic intervention.”

https://insight.jci.org/articles/view/139342 “Pathogenic and therapeutic role for NRF2 signaling in ultraviolet light–induced skin pigmentation”


Didn’t understand the 2nd experiment’s human dose of 5 nM sulforaphane. The lead author’s cited 2017 study Randomized, split-body, single-blinded clinical trial of topical broccoli sprout extract: Assessing the feasibility of its use in keratin-based disorders used “500 nmol of sulforaphane/mL.” Unless my math is off, the current study and previous study’s doses weren’t equivalent since 1 nM = 0.001 nmol/mL.

I’d like to know more about subjects who didn’t respond to topical sulforaphane treatment. What happened in their lives to make them dead to an evolutionarily-selected antioxidant and anti-inflammatory signaling pathway that influences many other internal environmental signals? Guess we’ll have to wait for:

“Further clinical studies with an increased number of human subjects, longer treatment regimens, and additional body sites are needed to further assess the long-term effects of NRF2 activation on photoaging.”

See Eat broccoli sprouts for your skin! and Eat broccoli sprouts for your hair! for similar studies.


Owl before sunrise

Week 28 of Changing to a youthful phenotype with broccoli sprouts

Did a little math to end this 28th week of eating a clinically relevant weight of microwaved broccoli sprouts every day:

  • I changed the title of weekly updates after Week 7 as a result of A rejuvenation therapy and sulforaphane. Numbers used from its study: “Rats were injected four times on alternate days for 8 days.”
  • Study numbers in Part 2 of Rejuvenation therapy and sulforaphane regarding the new human-rat relative biological age epigenetic clock: “The maximum lifespan for rats and humans were set to 3.8 years and 122.5 years, respectively.” I’m at a similar percentage of species maximum lifespan as were the study’s treated subjects.
  • A human-equivalent multiplication factor that can be applied to a rat post-development time period is 122.5 / 3.8 = 32.2. An 8-day rat treatment period ≈ 258 human days, and 258 / 7 ≈ 37 weeks.

To paraphrase the study’s lead laboratory researcher’s An environmental signaling paradigm of aging paper, aging is a programmed series of life stages. A body clock reset described there and subsequently experimentally tested changed 30 measurements to earlier life stages.

A reset may not require more than what I’ve been doing since the end of March. Maybe 28 weeks hasn’t been long enough to find out?


See the below discussion for a different point of view. I don’t think relative rates of metabolism between species would be more accurate than other measures because of individual differences among humans.

A chart from Microwave broccoli seeds to create sulforaphane of 10 people’s metabolisms after ingesting 200 μmol (35 mg) sulforaphane provides an example. Individual sulforaphane metabolites (DTC is dithiocarbamates) peak plasma measurements ranged from 0.359 μmol to 2.032 μmol.

sulforaphane peak plasma


So we’re patient.

Eat broccoli sprouts to pivot your internal environment’s signals

Two 2020 reviews covered some aspects of a broccoli sprouts primary action – NRF2 signaling pathway activation:

“Full understanding of the properties of drug candidates rely partly on the identification, validation, and use of biomarkers to optimize clinical applications. This review focuses on results from clinical trials with four agents known to target NRF2 signaling in preclinical studies, and evaluates the successes and limitations of biomarkers focused on:

  • Expression of NRF2 target genes [AKR1, GCL, GST, HMOX1, NQO1] and others [HDAC, HSP];
  • Inflammation [COX-2, CRP, IL-1β, IL-6, IP-10, MCP-1, MIG, NF-κB, TNF-α] and oxidative stress [8-OHdG, Cys/CySS, GSH/GSSG] biomarkers;
  • Carcinogen metabolism and adduct biomarkers in unavoidably exposed populations; and
  • Targeted and untargeted metabolomics [HDL, LDL, TG].

No biomarkers excel at defining pharmacodynamic actions in this setting.

SFN [sulforaphane] seems to affect multiple downstream pathways associated with anti-inflammatory actions. NRF2 signaling may be but one pivotal pathway.

SFN is generally considered to be the most potent natural product inducer of Nrf2 signaling. Studies in which these actions are diminished or abrogated in parallel experiments in Nrf2-disrupted mice provide the strongest lines of evidence for a key role of this transcription factor in its actions.

It is equally evident that other modes of action contribute to the molecular responses to SFN in animals and humans. Such polypharmacy may well contribute to the efficacy of the agent in disease prevention and mitigation, but obfuscates the value of specific pharmacodynamic biomarkers in the clinical development and evaluation of SFN.”

https://www.mdpi.com/2076-3921/9/8/716/htm “Current Landscape of NRF2 Biomarkers in Clinical Trials”


Why do researchers still not use epigenetic clocks in sulforaphane clinical trials? Forty mentions of disease in this review, but no consideration of aging?

This was another example of how researchers – even when stuck in a paradigm they know doesn’t sufficiently explain their area (“No biomarkers excel”) – don’t investigate other associated research areas. Why not?

Here’s what Part 2 of Rejuvenation therapy and sulforaphane had to say to those stuck on biomarkers:

“While clinical biomarkers have obvious advantages (being indicative of organ dysfunction or disease), they are neither sufficiently mechanistic nor proximal to fundamental mechanisms of aging to serve as indicators of them. It has long been recognized that epigenetic changes are one of several primary hallmarks of aging.

DNA methylation epigenetic clocks capture aspects of biological age.”


The second review Epigenetic Regulation of NRF2/KEAP1 by Phytochemicals also completely whiffed on epigenetic clocks. One mention of aging in this review, but it wasn’t of:

  • Citation 104 from Archives of Gerontology and Geriatrics; nor of
  • Citation 108 from the March 31, 2020, Aging journal; nor of
  • Citation 131 “Dietary epigenetics in cancer and aging.”

But epigenetic clock and aging associations were certainly in this review’s scope. For example, Citation 119 said:

“Nrf2 transcriptional activity declines with age, leading to age-related GSH loss among other losses associated with Nrf2-activated genes. This effect has implications, too, for decline in vascular function with age. Some of the age-related decline in function can be restored with Nrf2 activation by SFN.”

Why would people bother with phytochemicals (buzzword “compounds produced by plants”) unless to either ameliorate symptoms or address causes?

“Epigenetic Regulation of NRF2/KEAP1 by Phytochemicals” doesn’t occur in just laboratory situations. It’s also part of daily life.

These reviewers were straight-forward with side effects for two of the first review’s four items:

“The best known NRF2 activator that has obtained clinical approval is dimethyl fumarate for the treatment of multiple sclerosis. However, it has several side effects, including allergic reactions and gastrointestinal disturbance. There are a few related agents in clinical trials, such as Bardoxolone and SFX-01, a synthetic derivative of sulforaphane, which also exhibit less than desirable outcomes.”


Human relevance of rodent sulforaphane studies

After reading through findings of several dozen rodent studies this evening, I thought it would be worthwhile to revisit analysis of human relevance provided by one paper of How much sulforaphane is suitable for healthy people?

“Comparisons of published oral doses of sulforaphane administered to mice or rats and sulforaphane (tablets or sulforaphane-rich broccoli preparations) or glucoraphanin-rich broccoli preparations administered to humans.

The allometric scaling of the murine doses uses the correction factor of 0.081 and those for rat doses 0.162. Human doses were based on an estimate of 70 kg body weights in each study.”

A confession followed:

“Animal studies have not delivered all that might be expected of them. Pre-clinical experimentalists have not thought carefully about the selection of dose (or route) and its relevance to clinical utility.

Over two-thirds of the animal studies have used doses that exceed the highest (and bordering on intolerable) doses of sulforaphane used in humans.

Few studies have included a dose-response. The greater than 4-log spread of doses used in mice appears to be driven by needs for effect reporting in publications rather than optimization of translational science.

Authors of this review have contributed to this dose skewing.”


Let’s narrow this graphic to a human-relevant range:

human-rodent-relevant-dosages

48 of the 114 rodent study doses were in an allometric range applicable to humans.

Clinically relevant sulforaphane human doses start at a 100 µmol amount (17.73 mg). The graphic normalized human weights to 70 kg, so 100 µmol / 70 kg is 1.43 µmol / kg. Eyeballing the graphic, 43 of the 114 rodent study doses were in an allometric range applicable to human clinical doses.

But only three of the human sulforaphane study doses were above 4 µmol / kg. This indicator of the mentioned “intolerable doses” will limit clinically relevant oral doses to no more than 17.73 mg x (4 / 1.43) ≈ 50 mg in one serving.

Reviewing clinical trials of broccoli sprouts and their compounds described a sulforaphane study with doses above 4 µmol / kg:

“They proposed the intake of 15 capsules of broccoli sprouts at a time, giving 90 mg of SFN and 180 mg of glucoraphanin, a never before tested dosage, which was established because of the poor life expectancy of the patients and the aggressive characteristics of this type of cancer.

Secondary effects of the chemotherapy, the lack of appetite, nausea, vomits, diarrhea, mouth sores, etc., were factors that made it very difficult for the patients to intake 15 pills at once, as the study initially planned.

Progression of the pancreatic cancer and the GI symptomatology led to a high rate of drop-off of:

  • 72% in the treatment group; and
  • 55% abandonment in the control group!

Therefore, the results were not significant.”

Our model clinical trial Effects of long-term consumption of broccoli sprouts on inflammatory markers in overweight subjects calculated subjects’ mean weight in Table 1 as “85.8 ± 16.7 kg.” Its average glucoraphanin dose per kg body weight was 117 μmol / 85.8 kg = 1.36 μmol / kg.

Per Estimating daily consumption of broccoli sprout compounds, my twice-a-day consumption of a total 131 grams microwaved broccoli sprouts represents a worst-case 52 mg sulforaphane daily intake. This is ≈ 3 µmol / kg, the graphic’s second-largest sulforaphane amount cluster.


Only 9 of the 114 rodent studies were in an allometric range that was both:

  • Clinically relevant to humans as a lower boundary; and
  • Tolerable to humans as an upper boundary.

human-rodent clinically relevant tolerable dosages

The main purpose of animal studies is to help humans. Which researchers conducted sulforaphane studies that could actually help humans?

Grow a Victory Garden in mason jars

I tried a new process with success during the past 27th week of eating broccoli sprouts every day. My son suggested that mason jars with strainer lids would streamline the broccoli sprout production process. He was right, and then some.

I start a new batch every twelve hours. The left jar contained soaking seeds.

Here are thirteen measurements from this week compared with weights of a similar period last month. Starting amounts of broccoli seeds were all 10.7 grams, batches were rinsed three times each day on a 12 hour-6 hour-6 hour schedule, and weights taken at the 72-hour point:

Higher weights with less variation were reflected in broccoli sprout sizes. Few sprouts grew over one inch in three days when in bowls, but look at them now:

Larger broccoli sprouts taste better, too. After microwaving them on 1000W full power for 35 seconds to achieve up to but not exceeding 60°C (140°F), I wait five minutes to allow further myrosinase hydrolization of glucoraphanin and other glucosinolates into sulforaphane and other healthy compounds.

Further changes from what’s outlined in Step 5 of Grow a broccoli sprouts Victory Garden today! include:

  • I don’t shape batches anymore. I do fill each pint jar to the top and let it sit for five minutes in order to soak all seeds and sprouts.
  • I leave cooking water in after microwaving rather than straining it out. Although some leaching of water-soluble glucoraphanin may occur, I drink that water anyway.
  • I don’t mix in mustard, sauerkraut, or other flavorings. Still trying to make unsalted sauerkraut that tastes good.

I mistakenly pasted in a 9/10 p.m. value of 69.9 grams instead of its a.m. value of 66.0. Correcting it in my workbook changed the sample average from 68.8 g to 68.5 g. The correction didn’t change either the sample’s 4.9 g standard deviation value or the null hypothesis’ failed-test 0.0258 p-value.


This post is my one and only experiment with using the “new” retro Word Press block editor to start a new blog post. 😦 Word Press management knew this non-productive change was a non-starter, but foisted it on their users for their own convenience. 😦

They require me – along with hundreds of thousands of Word Press users – to edit blog posts with it. 😦 If retro is better, why don’t we all just go back eight decades to the most primitive text editor?

Treating psychopathological symptoms will somehow resolve causes?

This 2020 Swiss review subject was potential glutathione therapies for stress:

“We examine the available data supporting a role for GSH levels and antioxidant function in the brain in relation to anxiety and stress-related psychopathologies. Several promising compounds could raise GSH levels in the brain by either increasing the availability of its precursors or the expression of GSH-regulating enzymes through activation of Nrf2.

GSH is the main cellular antioxidant found in all mammalian tissues. In the brain, GSH homeostasis has an additional level of complexity in that the expression of GSH and GSH-related enzymes are not evenly distributed across all cell types, requiring the coordination between neurons and astrocytes to neutralize oxidative insults.

Increased energy demand in situations of chronic stress leads to mitochondrial ROS overproduction, oxidative damage and exhaustion of GSH pools in the brain.

Several compounds can function as precursors of GSH by acting as cysteine (Cys) donors such as taurine or glutamate (Glu) donors such as glutamine (Gln). Other compounds stimulate the synthesis and recycling of GSH through the activation of the Nrf2 pathway including sulforaphane and melatonin. Compounds such as acetyl-L-carnitine can increase GSH levels.”

https://www.sciencedirect.com/science/article/abs/pii/S0149763419311133 “Therapeutic potential of glutathione-enhancers in stress-related psychopathologies” (not freely available)


Many animal studies of “stress-related psychopathologies” were cited without noting applicability to humans. The reviewers instead had curious none-of-this-means-anything disclaimers like:

“Comparisons between studies investigating brain disorders of such different nature such as psychiatric disorders or neurodegenerative diseases, or even between brain or non-brain related disorders should be made with caution.”

Regardless, this paper had informative sections for my 27th week of eating broccoli sprouts every day.

1. I forgot to mention in Broccoli sprout synergies that I’ve taken 500 mg of trimethyl glycine (aka betaine) twice a day for over 15 years. Section 3.1.2 highlighted the amino acid glycine:

“Endogenous synthesis is insufficient to meet metabolic demands for most mammals (including humans) and additional glycine must be obtained from the diet. While most research has focused on increasing cysteine levels in the brain in order to drive GSH synthesis, glycine supplementation alone or in combination with cysteine-enhancing compounds are gaining attention for their ability to enhance GSH.”

2. The amino acid taurine dropped off my supplement regimen last year after taking 500 mg twice a day for years. It’s back on now after reading Section 3.1.3:

“Most studies that reported enhanced GSH in the brain following taurine treatment were performed under a chronic regimen and used in age-related disease models. Such positive effects of taurine on GSH levels may be explained by the fact that cysteine is the essential precursor to both metabolites, whereby taurine supplementation may drive the metabolism of cysteine towards GSH synthesis.

3. A study in Upgrade your brain’s switchboard with broccoli sprouts was cited for its potential:

“Thalamic GSH values significantly correlated with blood GSH levels, suggesting that peripheral GSH levels may be a marker of brain GSH content. Studies point to the capacity of sulforaphane to function both as a prophylactic against stress-induced behavioral changes and as a positive modulator in healthy animals.”


Sunrise minus 5 minutes