Eat broccoli sprouts for your eyes

This 2020 review subject concerned a leading cause of blindness:

“Advanced glycation end products (AGEs) are toxic compounds that have adverse effects on many tissues including the retina and lens. AGEs promote the formation of reactive oxygen species (ROS), which, in turn, boost the production of AGEs, a vicious cycle.

Diabetic retinopathy (DR) is a devastating microvascular complication of diabetes mellitus and the leading cause of blindness in working-age adults. The onset and development of DR is multifactorial. Lowering AGEs accumulation may represent a potential therapeutic approach.

Once AGEs are formed, most are irreversible. Cataracts are perhaps the earliest pathobiology of AGEs:

Nε-(carboxymethyl)-lysine (CML) [a representative AGE] in lens crystallins from diabetic (■) and non-diabetic (♦) subjects as a function of age.

The glyoxalase system is a protective mechanism that slows down synthesis of AGEs by limiting reactive dicarbonyls formed during sugar metabolism. Glutathione (GSH) in the eye is present at concentrations many times blood levels, and is a critical component of the glyoxalase system.

Proteomic analysis identified GLO1 [glyoxalase 1] as a protein differentially expressed in cells treated with sulforaphane. Sulforaphane inhibited AGEs-derived pericyte damage and delayed diabetes-induced retinal photoreceptor cell degeneration.

No AGE inhibitors have reached clinical use. The glyoxalase system and discovery of compounds that enhance this detoxifying activity represent a therapeutic alternative to fight glycation-derived damage.”

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


The above graph – plotting a cataract AGE level against chronological age – represented life stage progression without effective personal agency, without taking responsibility for your one precious life.

Citation 156 was Activation of Nrf2 attenuates carbonyl stress induced by methylglyoxal in human neuroblastoma cells: Increase in GSH levels is a critical event for the detoxification mechanism (not freely available):

“The present study focused on the methylglyoxal (MG) detoxification mechanism. MG treatment resulted in accumulation of modified proteins bearing the structure of AGEs.

This accumulation was suppressed by activation of the Nrf2 pathway prior to MG exposure via pre-treatment with an Nrf2 activator:

Although pre-treatment with the Nrf2 activator did not affect mRNA levels of GLO1, expressions of GCL and xCT mRNA, involved in GSH synthesis, were induced prior to increase in GSH levels.

These results indicated that increase in GSH levels promoted formation of the GLO1 substrate, thereby accelerating MG metabolism via the glyoxalase system and suppressing its toxicity. Promotion of GSH synthesis via the Nrf2/Keap1 pathway is important in MG detoxification.”

Continued in Part 2.


PXL_20201121_113656177

Nrf2 and Parkinson’s disease

This 2020 rodent study investigated a long non-coding RNA (lncRNA) in Parkinson’s disease:

“Knockdown of MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) lncRNA inhibited elevated nuclear factor (erythroid-derived 2)-like-2 factor (NRF2) expression, thereby inhibiting inflammasome activation and ROS (reactive oxygen species) production. MALAT1 was shown to promote neuroinflammation by recruiting enhancer of zeste homologue 2 (EZH2) to the promoter of NRF2, suppressing Nrf2 expression.

EZH2 catalyses generation of trimethylated H3K27 (H3K27me3) from histone H3 at lysine 27 (H3K27). EZH2 plays an important role in regulating the essential genes for inflammation in microglial activation, which induces neurodegeneration in the central nervous system.

Our results also validated MALAT1 binding to EZH2 in LPS-treated BV2 cells, which further recruited H3K27me3 to the gene promoter loci of Nrf2 to repress Nrf2 transcription. Although silencing MALAT1 did not alter global EZH2 expression levels, decreased binding between EZH2 and the Nrf2 promoter was observed. Previous studies have revealed that lncRNAs regulate the function of EZH2 in a similar manner.

MALAT1 epigenetically inhibits NRF2, thereby inducing inflammasome activation and ROS production in PD mouse and microglial cell models. To the best of our knowledge, it is first report of the important role of EZH2 in regulating the expression of Nrf2 to activate microglial inflammation.”

https://molecularbrain.biomedcentral.com/articles/10.1186/s13041-020-00656-8 “LncRNA MALAT1 facilitates inflammasome activation via epigenetic suppression of Nrf2 in Parkinson’s disease”


Eat broccoli sprouts today! referenced a letter to the editor that cited The Ezh2 Polycomb Group Protein Drives an Aggressive Phenotype in Melanoma Cancer Stem Cells and is a Target of Diet Derived Sulforaphane which found:

“SFN treatment is associated with reduced Ezh2 level and H3K27me3 formation.”

However, that study didn’t link sulforaphane’s main effect of Nrf2 signaling pathway activation to these specific treatment effects.

This post was inspired by our latest subscriber, Dr. Albert F. Wright, who is battling PD with – among other treatments – broccoli seeds.


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.

Epigenetic clock technology

This 2020 Norwegian study investigated current epigenetic clock technology:

“Epigenetic clocks are based on CpGs from the Illumina HumanMethylation450 BeadChip (450 K) which has been replaced by the latest platform, Illumina MethylationEPIC BeadChip (EPIC). EPIC is a major improvement over its predecessor, 450 K (> 450,000 CpGs), in terms of the number of probes (> 850,000 CpGs) and the genomic coverage of regulatory elements.

The training set of the other epigenetic clocks was mostly based on 450 K, except for the Horvath Skin & Blood clock which used both 450 K and EPIC-derived DNAm data. Additional CpGs on EPIC do not enhance the accuracy or precision of the epigenetic clocks when the training set is reduced.

We validated epigenetic clocks in EPIC-derived blood-based DNAm data (n = 470; 305 European women and 165 South Asian women). eABEC showed that the epigenetic age acceleration (EAA; residuals from the regression of DNAm age on chronological age) was higher in South Asian women than in Norwegian women.

The reason for the higher precision is likely due to the large training set (n = 2227) and the wide age-span of the samples (19 to 88 years for the training set of eABEC).

EPIC probes that are designed to cover regulatory regions did not increase precision. It is difficult to dismiss the possibility that other regulatory CpGs not currently included on EPIC might improve age prediction.”

https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-020-07168-8 “Blood-based epigenetic estimators of chronological age in human adults using DNA methylation data from the Illumina MethylationEPIC array”


The study’s main point was lacks in the current technology. The above graphic demonstrated that epigenetic clocks could do better across different ethnicities.

The study repeated a point from An epigenetic clock review by committee about increasing training set size. These researchers missed a point from Do epigenetic clocks measure causes or effects? that:

“The power of these measures as diagnostic and prognostic may stem from the use of longitudinal data in training them. Rather than continuing to train chronological age predictors using diverse data, it may be more advantageous to retrain some of the existing measures by predicting longitudinal outcomes.”

They also didn’t assign much relevance to coverage improvements of The epigenetic clock now includes skin:

“Although the skin-blood clock was derived from significantly less samples (~900) than Horvath’s clock (~8000 samples), it was found to more accurately predict chronological age, not only across fibroblasts and skin, but also across blood, buccal and saliva tissue.”


What I’d like to know about epigenetic clock measurements of biological age is: Why aren’t thousands of studies using them every year? How can we expect continuous improvements in their technologies or coverages or training sets without widespread use?

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

Nano-sulforaphane vs. barbecue chemicals

This 2020 chicken study investigated the capability of nano-sulforaphane to protect embryonic survival and neurogenesis from a barbecued meat chemical:

“Common teratogenic [of, relating to, or causing malformations of an embryo or a fetus] factors related to the development of the nervous system, such as alcohol consumption and smoking, have attracted wide attention. Teratogenic factors such as PhIP, the most abundant amine produced in common cooking procedures, can affect early embryonic development, leading to abnormal development of the nervous system.

Nano-sized medicine, in comparison with conventional medicine, leads to increased active concentrations and bioavailability. Both PhIP and nanoparticles can cross the placental barrier and enter the fetus from the external environment.

Chick embryos (100 per group) were incubated with 0.1% DMSO (Control); 20μM, 100μM, 200μM, or 300μM PhIP; or 200μM PhIP + 5μM Nano-SFN [sulforaphane] for 36 h:

  • Mortality rates were 0% for the Control, 8% with 20μM PhIP, 20% with 100μM PhIP, 53% with 200μM PhIP, 85% with 300μM PhIP, and 7% with 200μM PhIP + 5μM Nano-SFN.
  • Neural tube malformation rates [for the remaining live embryos] were 0% for the Control, 5% with 20μM PhIP, 14% with 100μM PhIP, 36% with 200μM PhIP, 14% with 300μM PhIP, and 6% with 200μM PhIP + 5μM Nano-SFN.

Women at the early stage of pregnancy should avoid barbecue. Instead, increase intake amount of cruciferous vegetables, which benefits fetal neural development.”

https://www.sciencedirect.com/science/article/abs/pii/S0940960220301618 “Nano-sulforaphane attenuates PhIP-induced early abnormal embryonic neuro-development” (not freely available)


PXL_20201022_225011002.NIGHT

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

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.”


Increasing carbon dioxide levels increases beneficial broccoli sprout compounds

This 2020 study used IPCC unscientific, politically-motivated, wild-ass guesses for year 2100 CO2 levels to find that broccoli sprouts – like most plants – benefit when CO2 is increased:

“Elevated CO2 (eCO2, 620 ppm, the expected IPCC-SRES B2-scenario prediction of eCO2 of the year 2100) was applied for 9 days to further improve nutritive and health-promoting values of three cultivars of broccoli sprouts.

  • eCO2 improved sprouts growth and induced GLs [glucosinolates] accumulation.
  • There were increases in myrosinase activity, which stimulated GLs hydrolysis to yield health-promoting sulforaphane.
  • Low levels of sulforaphane nitrile were detected and positively correlated with reduced epithiospecifier protein after eCO2 treatment.
  • High glucoraphanin and sulforaphane levels in eCO2 treated sprouts improved the anticarcinogenic and anti-inflammatory properties of their extracts.

In conclusion, eCO2 treatment enriches broccoli sprouts with health-promoting metabolites and bioactivities.”

https://www.sciencedirect.com/science/article/abs/pii/S030881462030964X “Elevated CO2 improves glucosinolate metabolism and stimulates anticancer and anti-inflammatory properties of broccoli sprouts” (not freely available)


This study was sponsored in Saudi Arabia. Would gathering such scientific evidence even be permitted in more “enlightened” countries?

Performing research on obvious lies and thinking for yourself isn’t allowed anymore in most “learning” institutions. You already knew that, didn’t you?

At the end of How much sulforaphane is suitable for healthy people? I applauded my high-school literature teachers for forcing their students to demonstrate that they could think for themselves. I didn’t mention that each monthly assignment to read two books, then compare-and-contrast them in a 3-page handwritten paper, was individualized so that students couldn’t undo the assignment’s purpose with parasitical collaboration.

This former practice remains a good measure of intentional dumbing-down of young people, the purpose of which has become clearer.

Jet fuel exposure causes diseases in the great-grand offspring

This 2020 Washington State University rodent study examined how great-grandmothers’ JP-8 exposures produced diseases in their great-grand offspring:

“Ancestral exposure to environmental influences such as toxicants, abnormal nutrition, and traumatic stress can affect the germline epigenome and promote the epigenetic transgenerational inheritance of adult onset disease in various organisms from plants to humans. Biological mechanisms underlying transgenerational epigenetic inheritance induced by jet fuel exposure are further investigated in the current study.

Genome-wide association studies (GWAS) have found specific genetic mutations associated with human pathologies, however these genetic mutations generally appear in less than 1% of the disease population. In contrast, epimutations (DNA methylation, histone modifications, non-coding RNA, chromatin structure, and RNA methylation alterations) seem to have a higher frequency and appear in more individuals with the diseases. Determining epigenetic biomarkers for these diseases could become especially useful indicators of environmental exposures and disease susceptibility in the human population.

The number of differential methylated regions (DMRs) found in the transgenerational F3 males is between 100 and 500 for each individual pathology. Few DMRs overlap between the different pathologies which supports the possible use of epimutations as biomarkers of disease. Although further studies are required, the lack of a subpopulation of DMRs overlapping with all pathologies suggests that at a more stringent statistical threshold there are not common DMRs among specific diseases.

Although females develop transgenerational disease, insufficient numbers of oocytes can be obtained on individuals to allow epigenetic associations to be assessed. The study only examined male pathology and associated sperm epimutation associations.”

https://www.sciencedirect.com/science/article/pii/S0890623820301982 “Epigenome-wide association study for transgenerational disease sperm epimutation biomarkers following ancestral exposure to jet fuel hydrocarbons”


The only associations these study subjects had with JP-8 were their great-grandmothers’ jet fuel exposures while pregnant with their grandparents. Other environmental toxicants studied by this group that produced similar transgenerationally inherited diseases were DDT, atrazine, and vinclozolin.

Ever think about your great-grandchildren?

Unraveling oxytocin – is it nature’s medicine?

This 2020 review attempted to consolidate thousands of research papers on oxytocin:

“Chemical properties of oxytocin make this molecule difficult to work with and to measure. Effects of oxytocin are context-dependent, sexually dimorphic, and altered by experience. Its relationship to a related hormone, vasopressin, have created challenges for its use as a therapeutic drug.

Widely used medical interventions i.e.:

  • Exogenous oxytocin, such as Pitocin given to facilitate labor;
  • Opioid medications that block the oxytocin system; or
  • Cesarean sections that alter exposure to endogenous oxytocin

have lasting consequences for the offspring and/or mother.

Such exposures hold the potential to have epigenetic effects on the oxytocin systems, including changes in DNA methylation. These changes in turn would have lasting effects on the expression of receptors for oxytocin, leaving individuals differentially able to respond to oxytocin and also possibly to the effects of vasopressin.

Regions with especially high levels of OXTR [oxytocin receptor gene] are:

  • Various parts of the amygdala;
  • Bed nucleus of the stria terminalis;
  • Nucleus accumbens;
  • Brainstem source nuclei for the autonomic nervous system;
  • Systems that regulate the HPA axis; as well as
  • Brainstem tissues involved in pain and social attention.

Oxytocin protects neural cells against hypoxic-ischemic conditions by:

  • Preserving mitochondrial function;
  • Reducing oxidative stress; and
  • Decreasing a chromatin protein that is released during inflammation

which can activate microglia through the receptor for advanced glycation end products (RAGE). RAGE acts as an oxytocin-binding protein facilitating the transport of oxytocin across the blood-brain barrier and through other tissues.

Directionality of this transport is 5–10 times higher from the blood to the brain, in comparison with brain to blood transport. Individual differences in RAGE could help to predict cellular access to oxytocin and might also facilitate access to oxytocin under conditions of stress or illness.

Oxytocin and vasopressin and their receptors are genetically variable, epigenetically regulated, and sensitive to stressors and diet across the lifespan. As one example, salt releases vasopressin and also oxytocin.

Nicotine is a potent regulator of vasopressin. Smoking, including prenatal exposure of a fetus, holds the potential to adjust this system with effects that likely differ between males and females and that may be transgenerational.

Relative concentrations of endogenous oxytocin and vasopressin in plasma were associated with:

These studies support the usefulness of measurements of both oxytocin and vasopressin but leave many empirical questions unresolved.

The vast majority of oxytocin in biosamples evades detection using conventional approaches to measurement.”

https://pharmrev.aspetjournals.org/content/pharmrev/72/4/829.full.pdf “Is Oxytocin Nature’s Medicine?”


I appreciated efforts to extract worthwhile oxytocin research from countless poorly performed studies, research that wasted resources, and research that actually detracted from science.

I was disappointed that at least one of the reviewers didn’t take this review as an opportunity to confess their previous wastes like three flimsy studies discussed in Using oxytocin receptor gene methylation to pursue an agenda.

Frank interpretations of one’s own study findings to acknowledge limitations is one way researchers can address items upfront that will be questioned anyway. Such analyses also indicate a goal to advance science.

Although these reviewers didn’t provide concrete answers to many questions, they highlighted promising research areas, such as:

  • Improved approaches to oxytocin measurements;
  • Prenatal epigenetic experience associations with oxytocin and OXTR; and
  • Possible transgenerational transmission of these prenatal epigenetic experiences.