384 replies to this topic

[dr_chaos]

http://www.fasebj.or...stract/22/3/703

Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways
Hani Atamna¹, Andy Nguyen, Carla Schultz, Kathleen Boyle, Justin Newberry, Hiroyuki Kato and Bruce N. Ames Nutrition and Metabolism Center, Children's Hospital Oakland Research Institute, Oakland, California, USA





¹Correspondence: Nutrition & Metabolism Center, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA, 94609-1673, USA. E-mail: hatamna@chori.org<script type="text/javascript">

Methylene blue (MB) has been used clinically for about a century to treat numerous ailments. We show that MB and other diaminophenothiazines extend the life span of human IMR90 fibroblasts in tissue culture by >20 population doubling (PDLs). MB delays senescence at nM levels in IMR90 by enhancing mitochondrial function. MB increases mitochondrial complex IV by 30%, enhances cellular oxygen consumption by 37–70%, increases heme synthesis, and reverses premature senescence caused by H₂O₂ or cadmium. MB also induces phase-2 antioxidant enzymes in hepG2 cells. Flavin-dependent enzymes are known to use NAD(P)H to reduce MB to leucomethylene blue (MBH₂), whereas cytochrome c reoxidizes MBH₂ to MB. Experiments on lysates from rat liver mitochondria suggest the ratio MB/cytochrome c is important for the protective actions of MB. We propose that the cellular senescence delay caused by MB is due to cycling between MB and MBH₂ in mitochondria, which may partly explain the increase in specific mitochondrial activities. Cycling of MB between oxidized and reduced forms may block oxidant production by mitochondria. Mitochondrial dysfunction and oxidative stress are thought to be key aberrations that lead to cellular senescence and aging. MB may be useful to delay mitochondrial dysfunction with aging and the decrease in complex IV in Alzheimer disease.—Atamna, H., Nguyen, A., Schultz, C., Boyle, K., Newberry, J., Kato, H., Ames, B. N. Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways.

So what do you think about that?

[DrHealth]

Yeah I heard about this last month. They are trialing a drug of methylene blue here in the uk to treat alzheimer's disease. It is called rember. More information can be found here:

good informative movie from the bbc http://news.bbc.co.u...lth/7532180.stm


Its really cheap and easy to get hold of aswel, a common lab chemical. Any healthy volenteers want to give it ago?

[kismet]

Don't know if you regularly visit the board over at mprize, just wanted to remind, Michael (who else?) posted about rember: http://mfoundation.o...ighlight=rember
However, I haven't seen any in vivo research on methylene blue's life extension capabilities. It may be good for AD and aging of the brain, though.

[AgeVivo]

What do you think about testing it in MPrize@ home?

[geddarkstorm]

Apparently there has been evidence of MB increasing life span in mice, but since we're taking about a much lower dose than that used for most medicinal purposes, it's been largely overlooked.

I take MB and have for about half a year. Considering it has also been shown, at lower than medicinal doses, in rats, to significantly increase learning of all types, and memory, I have to comment subjectively that that apparently has been the same for me. That is incredibly subjective, without stringent memory tests, and I did do a lot of mental exercising which recorded mental parameters (lumosity.com) a few months previously to taking MB, but none after.

Edited by geddarkstorm, 03 February 2009 - 05:03 AM.

[lynx]

Provepharm raises the specter of heavy metal contamination. Depending on the exact nature of the metal, this may or may not be a problem at the doses being considered here. I lean toward "not", but this isn't the sort of thing that I would want to leave to chance. MB has been used as a human pharmaceutical; would that suggest a clean source? Someone with access to an analytical lab could run a sample of MB for metals, but having this done at a contract lab would run into some money. Another concern, FWIW, is

MB also induces phase-2 antioxidant enzymes in hepG2 cells.

This could impact the bioavailability of other supplements, but might not be a problem at the low doses being discussed here.

If it is actually a phaseII inducer that would be good in my opinion, however, very frequently the term inducer is mistakenly used for "substrate".

[niner]

Provepharm raises the specter of heavy metal contamination. Depending on the exact nature of the metal, this may or may not be a problem at the doses being considered here. I lean toward "not", but this isn't the sort of thing that I would want to leave to chance. MB has been used as a human pharmaceutical; would that suggest a clean source? Someone with access to an analytical lab could run a sample of MB for metals, but having this done at a contract lab would run into some money. Another concern, FWIW, is

MB also induces phase-2 antioxidant enzymes in hepG2 cells.

This could impact the bioavailability of other supplements, but might not be a problem at the low doses being discussed here.

If it is actually a phaseII inducer that would be good in my opinion, however, very frequently the term inducer is mistakenly used for "substrate".

A phase II inducer might be good if you are exposed to carcinogenic hydrocarbons on a regular basis, but if, like most of us, you aren't, then it might be a negative in that it would lead to quicker clearance of a lot of beneficial phytochemicals such as polyphenols, flavanols, etc. People take things like bioperine and quercetin in order to inhibit phase II enzymes. The confusion between inducers and substrates might derive from the fact that many substrates are inducers of the enzyme than metabolizes them. That is a sensible response, since if you have a lot of some xenobiotic chemical exposure, you will want more of the enzyme that gets rid of it.

[lynx]

Provepharm raises the specter of heavy metal contamination. Depending on the exact nature of the metal, this may or may not be a problem at the doses being considered here. I lean toward "not", but this isn't the sort of thing that I would want to leave to chance. MB has been used as a human pharmaceutical; would that suggest a clean source? Someone with access to an analytical lab could run a sample of MB for metals, but having this done at a contract lab would run into some money. Another concern, FWIW, is

MB also induces phase-2 antioxidant enzymes in hepG2 cells.

This could impact the bioavailability of other supplements, but might not be a problem at the low doses being discussed here.

If it is actually a phaseII inducer that would be good in my opinion, however, very frequently the term inducer is mistakenly used for "substrate".

A phase II inducer might be good if you are exposed to carcinogenic hydrocarbons on a regular basis, but if, like most of us, you aren't, then it might be a negative in that it would lead to quicker clearance of a lot of beneficial phytochemicals such as polyphenols, flavanols, etc. People take things like bioperine and quercetin in order to inhibit phase II enzymes. The confusion between inducers and substrates might derive from the fact that many substrates are inducers of the enzyme than metabolizes them. That is a sensible response, since if you have a lot of some xenobiotic chemical exposure, you will want more of the enzyme that gets rid of it.

Since fertility rates, sperm motility, testosterone levels have all been dropping like crazy, I assume that everyone has significant exposure.

Edited by lynx, 06 February 2009 - 06:54 PM.

[zawy]

There are U.K. researchers that shine red light through skin to cause MB to degrade(?) to a byproduct that kills cancer cells. As I recall, it worked pretty good as long as the tumor wasn't more than 1 or 2 inches beneath the skin (where the light can't go). I think they used 660 nm LEDs.

[zawy]

Here's an interesting article on MB and red light to treat cancer. It seems like this idea goes back to 1989.

http://cancerres.aac...ull/63/4/776#B8

"Treatment of cancer by PDT (photodynamic light therapy) usually involves the generation of reactive, excited oxygen molecules (1) A photosensitizing dye absorbs light, exciting it to an energetic triplet state capable of transferring its energy efficiently to ground state, triplet molecular oxygen to form excited, singlet oxygen. .... Because red light penetrates tissue much deeper than that of shorter wavelength, photosensitizers absorbing in the red are preferred. Phenothiazinium dyes such as MB⁺ or TB⁺ absorb strongly at approximately 600–670 nm, and have been identified as having potential for cancer diagnosis or treatment. ..... MB⁺ has been extensively investigated as a photocatalyst of oxidative processes, in experimental PDT in vitro and in vivo, and in limited clinical studies involving PDT (8,9) . We hypothesized that these dyes could be photocatalysts of oxidation of IAA, the commonest plant auxin (growth hormone), the dye triplet state oxidizing IAA to produce a radical-cation (Fig. 1) ....We have shown that products of IAA oxidation are cytotoxic to mammalian cells, including human tumor cells. Thus, the combination of IAA or suitable analogues with these photosensitizers could provide an alternative route to photosensitized cell killing not involving singlet oxygen. ... We show here that IAA, a common, nontoxic plant chemical, is transformed into potent cytotoxins using red light and these phenothiazinium dyes that are already in clinical use. Activating IAA rather than oxygen to form cytotoxins offers the potential to treat hypoxic tumors effectively.

In conclusion, the present results show that the phototoxicity of MB⁺ and TB⁺ is enhanced dramatically by the presence of IAA at concentrations likely to be achievable in humans. The use of IAA, or analogues with modified redox properties, with these or other suitable photosensitizers is attractive because of the opportunity for rational selection of both photosensitizer and IAA analogue, based on thermodynamic (redox) and kinetic arguments. In principle, a requirement is that the reduction potential of the photosensitizer triplet state [e.g., of the couple MB⁺/MB^·, where MB^· is the radical (semiquinone)] is higher than that of the indolyl radical cation (IAA^·+/IAA); ideally, the reduced dye should also be efficiently "redox cycled" to the parent dye by oxygen. These requirements appear to be fulfilled for MB⁺ and IAA."

I imagine light sticks being for throat and prostate cancer.

and it seems like if you take MB and get a lot of sun light it could kill viruses in the blood: "An increasingly important medical use of MB⁺ exploiting its photodynamic activity is to reduce viral contamination of blood plasma by MB⁺ and light."

"The prevalence of West Nile virus (WNV) infections and associated morbidity has accelerated in recent years. Of particular concern is the recent demonstration that this virus can be transmitted by blood products and can cause severe illness and mortality in transfusion recipients. We have evaluated methylene blue (MB) + light as a safe and cost-effective means to inactivate WNV in vitro. This regimen inactivated WNV with an IC₅₀ of 0.10 μM. Up to 10⁷ pfu/ml of WNV could be inactivated by MB + light with no residual infectivity. MB + light inactivated three primary WNV isolates from the years 1999, 2002 and 2003 and prevented mortality in a murine model for WNV infection. Since MB is already approved for human use at a dose of 100 mg/kg/day, we conjecture that MB + light treatment of blood products for high-risk patients will be efficacious and suitable for use in resource-limited settings."

Edited by zawy, 19 February 2009 - 01:24 PM.

[krillin]

PMID: 17721552 IC50 for MAO A inhibition is 164 nM: an order of magnitude lower than that needed to prevent tau filament formation.

PMID: 6603875 The photosensitizer effect can cause retinal damage if inadequate antioxidants are present.

Patent 7,335,505 Reduced methylene blue (leucomethylene blue) is more effective and crosses the blood brain barrier better. One recipe for reduction is 2 - 2.5 mg vitamin C per mg MB for three hours. IC50 for inhibiting tau filament formation is 4 microM.

PMID: 15611092 Myricetin is actually better than MB for inhibiting tau filament formation (IC50s of 1.2 and 1.9 microM). I had no luck finding any myricetin pharmacokinetics.

PMID: 14666245 Myricetin probably needs something like a lecithin liposome to cross the blood brain barrier. (Quercetin does.) Let's badger Source Naturals into providing this formulation, since their myricetin product is a tablet. (With a very nice banana smell.)

TauRx's second generation drug is called LMT-X. My guess from the name is that it's a shelf-stable leucomethylene blue formulation.

[geddarkstorm]

PMID: 17721552 IC50 for MAO A inhibition is 164 nM: an order of magnitude lower than that needed to prevent tau filament formation.

PMID: 6603875 The photosensitizer effect can cause retinal damage if inadequate antioxidants are present.

Patent 7,335,505 Reduced methylene blue (leucomethylene blue) is more effective and crosses the blood brain barrier better. One recipe for reduction is 2 - 2.5 mg vitamin C per mg MB for three hours. IC50 for inhibiting tau filament formation is 4 microM.

PMID: 15611092 Myricetin is actually better than MB for inhibiting tau filament formation (IC50s of 1.2 and 1.9 microM). I had no luck finding any myricetin pharmacokinetics.

PMID: 14666245 Myricetin probably needs something like a lecithin liposome to cross the blood brain barrier. (Quercetin does.) Let's badger Source Naturals into providing this formulation, since their myricetin product is a tablet. (With a very nice banana smell.)

TauRx's second generation drug is called LMT-X. My guess from the name is that it's a shelf-stable leucomethylene blue formulation.

That's great stuff. Of course, we aren't talking about using MB as a tau inhibitor, but a mitochondrial dysfunction reverser - which requires 100nM and starts to drop off at concentrations higher than that, so it seems. It also takes a high amount of MB to raise to the levels capable of being a photosynthesizer, and this switch is an all or nothing thing: that is, MB is not a photosensitizer below a critical concentration. It is pretty dang potent against cancer once it's past that point, when hit with a powerful enough light (obviously, sunlight cannot activate MB photosensitization).

[krillin]

It also takes a high amount of MB to raise to the levels capable of being a photosynthesizer, and this switch is an all or nothing thing: that is, MB is not a photosensitizer below a critical concentration. It is pretty dang potent against cancer once it's past that point, when hit with a powerful enough light (obviously, sunlight cannot activate MB photosensitization).

Neurons can be much more fragile than tumors, with nanomolar concentrations of sensitizers being effective in the first reference below. Google (methylene blue sunlight coliform) for examples of sunlight being enough to activate it, and also see the solar simulator experiment in the second reference below. Damage would probably be very slow to show up in vivo because of antioxidants, but I'm planning on living forever and want to save money on retina replacements. The MAOI effect, however, is the real killer for me, because I can't even tolerate the serotonin release caused by baclofen.

Izv Akad Nauk Ser Biol. 2000 Mar-Apr;(2):230-8.
[Study of the photodynamic effect of new photosensitizers on the single neuron]
Uzdenskiĭ AB, Zhavoronkova AA, Mironov AF, Kuz'min SG.
Rostov State University, Rostov-on-Don, Russia. uzd@krinc.ru

We studied reactions of isolated crayfish mechanoreceptor neurons to photodynamic effects of various photosensitizers: methylene blue, chlorins e6 and p6, sulfated allumophthalocyanin Photosens, Janus green B, protoporphyrin IX, and two derivatives of hematoporphyrin IX, Photoheme and Photosan-3. The neurons were irradiated by a helium-neon laser (632.8 nm, 0.3 Wt/cm3) after 30-min photosensitization. They proved to be very sensitive to the photodynamic effect: When the cells stained by photosensitizers at nanomolar concentrations were irradiated, their firing activity underwent irreversible changes and they died. The dynamics of the firing activity of the neurons depended on the photosensitizer type and concentration. Photosens, Photoheme and chlorin p6 proved to be the most efficient.

PMID: 10780116

Acta Microbiol Pol. 2003;52(1):65-79.
Study of solar photosensitization processes on dermatophytic fungi.
Ouf SA, Abdel-Kader MH, Shokeir HA, El-Adly AA.
Botany Department, Faculty of Science, Cairo University, Giza 12613, Egypt.

The antifungal activity of solar simulator was evaluated in presence of haematoporphyrin derivative (HPD), methylene blue (MB) and toluidine blue O (TBO) as photosensitizers. Seven dermatophytes were used as test fungi. The solar simulator at fluence rate 400 W/m2 for 30 minutes induced marked inhibition for spore germination of the photosensitized fungi. The rate of inhibition varied according to the fungal species and concentration of the photosensitizer. There was an increase in percentage inhibition of spore germination as the concentration of HPD or MB increased. Complete inhibition for spore germination of Trichophyton. verrucosum, T. mentagrophytes, and Miccrosporum canis was induced when these species were pretreated with 10(-3) M of HPD or MB before irradiation. Epidermophyton floccosum, T. rubrum, M. gypseum and T. violaceum were less sensitive to irradiation when pretreated with HPD or MB. On contrary, the maximum reduction in percentage spore germination was induced at the lowest concentration (10(-7) M) of TBO. The tested dermatophytes were mostly capable of producing different enzymes (keratinase, phosphatases, amylase, lipase). The separate application of radiation or photosensitizer was ineffective or exerted slight inhibition on enzyme production. However, the activity of the enzymes was drastically inhibited when the fungi were irradiated after their treatment with photosensitizer. T. verrucosum and T. mentagrophytes were the most sensitive. In a trail to apply a control measure against dermatomycosis using solar simulator radiation, the results revealed that the radiation was successful in curing the MB-photosensitized guinea pigs, artificially infected with T. verrucosum, T. mentagrophytes or M. canis. The percentage of recovery reached 100% in some treatments.

PMID: 12916729

[geddarkstorm]

This photosensitization thing seems quite absurd, to me anyways. MB isn't something brand new. It's been around for ages, used at concentrations way beyond 100nM (~1mg!) in humans and no ill "photosensitization" effects have been reported to my knowledge. Considering ~200mg of MB is used routinely for methylhemoglobinemia, and anywhere from ~733mg (12mg/kg/day) to ~1,500mg (24mg/kg/day) in humans is used to treat malaria with few ill effects (the minimal dosage needed to see any toxic effect what so ever from MB is estimated around 600mg from the rat toxicological studies)... Yeah, I'm sorry, but these studies with crayfish neurons (sunlight is actually 137 mW/cm2 at nigh noon, staring directly into the sun; so the crayfish study used three times the amount of energy that sunlight has, and you already can't stare directly into the sun, MB notwithstanding) and fungi (which used mM amounts of MB in their study) just don't cut it for me. Also, do we even know what doses were used in PMID: 6603875? They did say that vitamin E completely stopped any photosensitization by MB, and considering the issue is singlet oxygen production, which is made constantly all the time in our bodies, including our eyes, I really don't worry - the human body is quite apt at not only dealing with but actually using super oxide for a variety of signals.

I have no doubt, and this is only my opinion, that you have nothing to worry about with your retina and MB at these doses, what so ever. Not to mention humans have a better antioxidant system than most creatures on this planet, and even many mammals, our retinas are not prone to failure by any direct "sensitization" effect, and retinas can heal quite well. It's only if you lose the optic nerve or degenerate the retina faster than it can heal (usually this is genetic) that you run into trouble; and I'm afraid MB at these dosages just isn't going to be able to do that. You'll lose your lens long before retina trouble, more likely than not, since the lens is one of the very rare things that aren't turned over.

On the other hand, the MAO issue is not one to brush off or take lightly, especially if you have sensitivity to it that is high enough where even such a miniscule amount of MB could affect you (since it is going to be about 1/3rd inhibitory at 100nM, theoretically, depending on BBB crossing). Also, G6PD deficiency must never be crossed with MB, so that's something to keep in mind.

Edited by geddarkstorm, 26 February 2009 - 02:31 AM.

[krillin]

This photosensitization thing seems quite absurd, to me anyways. MB isn't something brand new. It's been around for ages, used at concentrations way beyond 100nM (~1mg!) in humans and no ill "photosensitization" effects have been reported to my knowledge. Considering ~200mg of MB is used routinely for methylhemoglobinemia, and anywhere from 2,200mg (36mg/kg) to 4,500mg (72mg/kg) in humans is used to treat malaria with few ill effects (the minimal dosage needed to see any toxic effect what so ever from MB is estimated around 600mg from the rat toxicological studies)... Yeah, I'm sorry, but these studies with crayfish neurons and fungi just don't cut it for me. Also, do we even know what doses were used in PMID: 6603875? They did say that vitamin E completely stopped any photosensitization by MB, and considering the issue is singlet oxygen production, which is made constantly all the time in our bodies, including our eyes, I really don't worry - the human body is quite apt at not only dealing with but actually using super oxide for a variety of signals.

I have no doubt, and this is only my opinion, that you have nothing to worry about with your retina and MB at these doses, what so ever. Not to mention humans have a better antioxidant system than most creatures on this planet, and even many mammals, our retinas are not prone to failure by any direct "sensitization" effect, and retinas can heal quite well. It's only if you lose the optic nerve or degenerate the retina faster than it can heal (usually this is genetic) that you run into trouble; and I'm afraid MB at these dosages just isn't going to be able to do that. You'll lose your lens long before retina trouble, more likely than not, since the lens is one of the very rare things that aren't turned over.

Why do we take lutein to prevent macular degeneration if our retinas are as robust as you claim? Do we have enough long-term users of MB to do an epidemiology study? Would any grant officer fund such a study if we did?

Your credibility would improve if you would do a cursory search before making absolute statements. I've already shown how unsubstantiated your "It also takes a high amount of MB to raise to the levels capable of being a photosynthesizer" and "obviously, sunlight cannot activate MB photosensitization" claims were. Now see how easy it is to knock down "our retinas are not prone to failure by any direct "sensitization" effect, and retinas can heal quite well".

J Biol Chem. 1995 Aug 11;270(32):18825-30.
Blue light-induced reactivity of retinal age pigment. In vitro generation of oxygen-reactive species.
Rózanowska M, Jarvis-Evans J, Korytowski W, Boulton ME, Burke JM, Sarna T.
Department of Biophysics, Jagiellonian University, Kraków, Poland.

Exposure of the eye to intense light, particularly blue light, can cause irreversible, oxygen-dependent damage to the retina. However, no key chromophores that trigger such photooxidative processes have been identified yet. We have found that illumination of human retinal pigment epithelium (RPE) cells with light induces significant uptake of oxygen that is both wavelength- and age-dependent. Analysis of photoreactivity of RPE cells and their age pigment lipofuscin indicates that the observed photoreactivity in RPE cells is primarily due to the presence of lipofuscin, which, under aerobic conditions, generates several oxygen-reactive species including singlet oxygen, superoxide anion, and hydrogen peroxide. We have also found that lipofuscin-photosensitized aerobic reactions lead to enhanced lipid peroxidation as measured by accumulation of lipid hydroperoxides and malondialdehyde in illuminated pigment granules. Hydrogen peroxide is only a minor product of aerobic photoexcitation of lipofuscin. We postulate that lipofuscin is a potential photosensitizer that may increase the risk of retinal photodamage and contribute to the development of age-related maculopathy.

PMID: 7642534

[geddarkstorm]

This photosensitization thing seems quite absurd, to me anyways. MB isn't something brand new. It's been around for ages, used at concentrations way beyond 100nM (~1mg!) in humans and no ill "photosensitization" effects have been reported to my knowledge. Considering ~200mg of MB is used routinely for methylhemoglobinemia, and anywhere from 2,200mg (36mg/kg) to 4,500mg (72mg/kg) in humans is used to treat malaria with few ill effects (the minimal dosage needed to see any toxic effect what so ever from MB is estimated around 600mg from the rat toxicological studies)... Yeah, I'm sorry, but these studies with crayfish neurons and fungi just don't cut it for me. Also, do we even know what doses were used in PMID: 6603875? They did say that vitamin E completely stopped any photosensitization by MB, and considering the issue is singlet oxygen production, which is made constantly all the time in our bodies, including our eyes, I really don't worry - the human body is quite apt at not only dealing with but actually using super oxide for a variety of signals.

I have no doubt, and this is only my opinion, that you have nothing to worry about with your retina and MB at these doses, what so ever. Not to mention humans have a better antioxidant system than most creatures on this planet, and even many mammals, our retinas are not prone to failure by any direct "sensitization" effect, and retinas can heal quite well. It's only if you lose the optic nerve or degenerate the retina faster than it can heal (usually this is genetic) that you run into trouble; and I'm afraid MB at these dosages just isn't going to be able to do that. You'll lose your lens long before retina trouble, more likely than not, since the lens is one of the very rare things that aren't turned over.

Why do we take lutein to prevent macular degeneration if our retinas are as robust as you claim? Do we have enough long-term users of MB to do an epidemiology study? Would any grant officer fund such a study if we did?

Your credibility would improve if you would do a cursory search before making absolute statements. I've already shown how unsubstantiated your "It also takes a high amount of MB to raise to the levels capable of being a photosynthesizer" and "obviously, sunlight cannot activate MB photosensitization" claims were. Now see how easy it is to knock down "our retinas are not prone to failure by any direct "sensitization" effect, and retinas can heal quite well".

J Biol Chem. 1995 Aug 11;270(32):18825-30.
Blue light-induced reactivity of retinal age pigment. In vitro generation of oxygen-reactive species.
Rózanowska M, Jarvis-Evans J, Korytowski W, Boulton ME, Burke JM, Sarna T.
Department of Biophysics, Jagiellonian University, Kraków, Poland.

Exposure of the eye to intense light, particularly blue light, can cause irreversible, oxygen-dependent damage to the retina. However, no key chromophores that trigger such photooxidative processes have been identified yet. We have found that illumination of human retinal pigment epithelium (RPE) cells with light induces significant uptake of oxygen that is both wavelength- and age-dependent. Analysis of photoreactivity of RPE cells and their age pigment lipofuscin indicates that the observed photoreactivity in RPE cells is primarily due to the presence of lipofuscin, which, under aerobic conditions, generates several oxygen-reactive species including singlet oxygen, superoxide anion, and hydrogen peroxide. We have also found that lipofuscin-photosensitized aerobic reactions lead to enhanced lipid peroxidation as measured by accumulation of lipid hydroperoxides and malondialdehyde in illuminated pigment granules. Hydrogen peroxide is only a minor product of aerobic photoexcitation of lipofuscin. We postulate that lipofuscin is a potential photosensitizer that may increase the risk of retinal photodamage and contribute to the development of age-related maculopathy.

PMID: 7642534

And I did edit my post some time before you made yours to include data that showed that the crayfish study was using three times that of actual sunlight energy. Not to mention, you don't look into the sun directly, do you? We only look at light reflected off objects, don't we?

Moreover retinas do heal, as retina based operations are done. Look at here (PMID: 18835472), and here where they did direct damage to the retina with lasers and watched healing occur. It is retina detachment from the underlying macula that is most dangerous.

So far you haven't knocked down anything I said. And I already posted a paper that showed clearly that MB is not a photosensitizer below a certain concentration, it has to be above a certain threshold to have any photosensitizing activity, but once it does cross this treshhold it is quite potent - whatever keeps it within ratio with natural antioxidant systems it would appear.

Even more interesting is those who take MB for genetic methylhemoglobinemia, they do have to take it over the long term. We don't hear of trouble with them, do we? I see nothing that knocks down my statements so far. If you can bring proof, of course I'll side with it, but all my searching shows no worries at such incredibly low concentrations of MB as 100nM when it comes to photosensitization.

EDIT: It seems MB has already been looked at specifically for if it damages rabbit eyes, and apparently no damaging effects where found at lower concentrations with direct injection (no changes to electrical signaling by the retinas were seen at any concentration of MB that they used, but some vacuolar changes happened at the higher doses which implies some toxicity). See here.

EDIT2: MB actually protects retina from oxidative damage and rotenone in this study (PMID: 16464752).

Edited by geddarkstorm, 26 February 2009 - 03:23 AM.

[niner]

Krillin! Good to see you back. This might put you at more ease on the photosensitization aspect: The spectral energy density at sea level of visible sunlight is about 1.3 W/m**2 / nm, where nm is the width of the wavelength band. MB has a broad absorption band about 100nm wide, from about 590 to 690nm. That would give you an energy of 130 microwatts/cm**2, assuming full absorbance, which is undoubtedly an overstatement. The laser used in the experiment was about 2,400 times more intense than this. FWIW, anyway.

[krillin]

And I did edit my post some time before you made yours to include data that showed that the crayfish study was using three times that of actual sunlight energy. Not to mention, you don't look into the sun directly, do you? We only look at light reflected off objects, don't we?

Moreover retinas do heal, as retina based operations are done. Look at here (PMID: 18835472), and here where they did direct damage to the retina with lasers and watched healing occur. It is retina detachment from the underlying macula that is most dangerous.

I knew the laser was way more powerful than sunlight, and I never said that retinas have no capacity to heal. Why do you think I wrote "Damage would probably be very slow to show up"? The indisputable facts are that

1. MB can generate ROS at nanomolar concentrations.
2. MB can generate ROS at solar intensities.
3. Our antioxidant defenses and healing capacity are inadequate even without the added burden of MB, or else macular degeneration wouldn't be such a common problem.

It's reckless to quibble that the fraction of solar radiation that makes it into the eye might not cause MB to generate any ROS. Whatever happened to the principle of presumed guilty before proven innocent?

So far you haven't knocked down anything I said.

An example. You said "our retinas are not prone to failure by any direct "sensitization" effect". That's exactly what lipofuscin does. You're obviously an intelligent person, so you're either not precisely conveying your ideas or are BSing us for some reason.

Even more interesting is those who take MB for genetic methylhemoglobinemia, they do have to take it over the long term. We don't hear of trouble with them, do we?

Why are you assuming that we would have heard of a problem if it existed? Say you're a doctor and a MB-using patient of yours gets macular degeneration when he's old. Old people get that all the time. Why bother reporting it? It's best not to assume that absence of evidence is evidence of absence.

Basically, what we have here is a finite risk for taking something that hasn't been documented to extend lifespan, or even square the curve. All I could find was this study on flies, which doesn't count because their antioxidant defenses suck.

Mech Ageing Dev. 1993 May;68(1-3):175-82.
Influence of photosensitizers and light on the life span of Drosophila.
Massie HR, Aiello VR, Williams TR.
Masonic Medical Research Laboratory, Utica, NY 13501.

The life span of adult Drosophila melanogaster fruit flies changed when they were fed two different photosensitizers. Methylene blue decreased the median life span by 49% when present in the food at a concentration of 0.001 M. Another photosensitizer, riboflavin, produced no changes in life span under the same conditions of a 12:12 h light/dark cycle at a daytime light intensity of 1000 lux. Flies exposed to constant darkness lived 43.2% longer than those exposed to constant light at a light intensity of 2000 lux. Under these conditions, riboflavin increased the life span of the flies exposed to constant light by as much as 25%. We conclude that riboflavin confers some degree of protection against the effects of constant light exposure. The completely different results obtained with riboflavin and methylene blue suggest a possible mechanism for photoageing involving photodynamic action mediated through the production of singlet oxygen.

PMID: 8350657

[geddarkstorm]

I knew the laser was way more powerful than sunlight, and I never said that retinas have no capacity to heal. Why do you think I wrote "Damage would probably be very slow to show up"? The indisputable facts are that

1. MB can generate ROS at nanomolar concentrations.

When hit with a powerful enough, proper wave length, light source - but not before then. In fact, it's documented as an antioxidant under most cellular conditions. Like all antioxidants, too much of it becomes pro-oxidant. Here's yet another example of MB being a protector of neurons from oxidative damage. Oh, and look, yet another example where MB attenuates super oxide production. So no, MB is NOT a ROS producer at nanomolar levels without sufficient light energy stimulation, but a potent antioxidant. To reiterate once again: MB seems to be a pretty dang potent antioxidant within the ranges we're talking about using (100-1000nM) for mitochondria stimulation and increased brain function, and even at the medicinal levels (which is 200 to 1000 times higher).

2. MB can generate ROS at solar intensities.

No. You have shown no proof of that, at all. I have seen no proof of that, at all. Rather, only counter proof. The crayfish paper used at least three times more energy than you can ever get from the sun (or they used 2,400 times that energy at MBs absorption spectrum according to Niner's calculation), even at noon at the equator, where sea level solar spectral energy maxes out at 137mW/cm^2. Now, the actual wavelengths that MB absorbs are the lowest energy wavelengths of visible light towards near infrared (590-690nm). And when MB was investigated directly with injections into the eyes of rabbits, at levels (in the eye, for all their doses) far beyond what you could get from taking 1mg a day, or even beyond at medical human dosages, they did not see any loss of retina function. There were signs of some toxicity at the higher dosages however.

3. Our antioxidant defenses and healing capacity are inadequate even without the added burden of MB, or else macular degeneration wouldn't be such a common problem.

Common problem? Age related macular degeneration is the highest cause leading to legal blindness with age, yes. However, it's estimated that only 10 percent of the population gets it over the age of 55, and up to 30 percent over the age of 75. The large majority of people do not get it. It would depend on what you classify as common if this is common or not. Furthermore, it is genetically caused predominately, not related to oxidative stress (non-carriers of the risk gene only have a 22% chance of macular degeneration by age 95). So no, our antioxidant systems are quite good enough for the vast majority of people.

It's reckless to quibble that the fraction of solar radiation that makes it into the eye might not cause MB to generate any ROS. Whatever happened to the principle of presumed guilty before proven innocent?

It is not reckless at all, but the very heart of the manner. MB isn't going to be generating ROS without sufficient light energy of the proper light spectrum getting to it - that is, it does not generate ANY ROS at all until it hits a certain light energy threshold, as a paper I already linked showed. That means that the proper amount of light has to filter through all the tissues and, if not directly entering the eye, bone. The only way to generate the highest amount of light energy onto the retina is by looking at a light source directly. This happens rarely (solar UV would do far more damage faster anyways), and we already have seen that actual sunlight doesn't carry the proper energy at all, even at high noon at the equator of the planet (where sunlight energy is its highest at sea level from anywhere on the planet).

On another note, MB can generate H2O2 ROS, but not super oxide, if you throw in a larger amount of it (mid micromols to low millimols), whereby it starts to interact with reductases, far more specifically with malaria versions than human versions. But this is completely different than "photosensitization".

By the way, It is also possible to create a false dilemma by, and mascaraing as, being overly cautious.

So far you haven't knocked down anything I said.

An example. You said "our retinas are not prone to failure by any direct "sensitization" effect". That's exactly what lipofuscin does. You're obviously an intelligent person, so you're either not precisely conveying your ideas or are BSing us for some reason.

Erm, lipofuscin is a byproduct of the light reaction, not an exogenous "sensitizer" in the sense that we were talking about with MB.

To clarify, lipofuscin doesn't seem to directly absorb light and generate super oxide like MB can after a certain threshold. Rather, lipofuscin impairs the clearance and cycling of retinal, and at the same time builds up retinyl palmitate that, once oxidized by light, becomes anhydroretinol, a signaling molecule which kicks on the apoptosis signaling cascade that then later goes on to generates ROS as part of its cascading signal. That is, lipofuscin, generated by the normal vision cycle over time, acts to impair the recycling of vision cycle compounds (trans-retinal), and builds up photosensitive byproducts that become signalling molecules to induce apoptosis. This is different from MB directly generating ROS and being an exogenous factor, not a byproduct of the vision cycle.

So, I don't think these two are comparable at all. And what's more, is I said "not prone to", not that it doesn't happen. Since around only 1/3rd of people ever get macular degeneration, and most of it is linked to certain genes, not "sensitization" for the generation of ROS and damage derived there in... I think I'm completely accurate in saying "not prone to failure by any direct "sensitization" effect".

On a personal note, I certainly haven't run around sounding pretentious and saying you are "BSing", even while proving some of your points unfounded and false.

Even more interesting is those who take MB for genetic methylhemoglobinemia, they do have to take it over the long term. We don't hear of trouble with them, do we?

Why are you assuming that we would have heard of a problem if it existed? Say you're a doctor and a MB-using patient of yours gets macular degeneration when he's old. Old people get that all the time. Why bother reporting it? It's best not to assume that absence of evidence is evidence of absence.

I believe that if their rates went up significantly, someone would have noticed that after all this time - as we've had plenty of time to notice such changed rates. Perhaps I just have more faith in medical and scientific observations. They got us this far, so far, after all.

Basically, what we have here is a finite risk for taking something that hasn't been documented to extend lifespan, or even square the curve. All I could find was this study on flies, which doesn't count because their antioxidant defenses suck.

I'm well aware of that study in flies, and they used a comparatively huge amount of MB, 1 mM in their food. Since the average person consumes around 4.7lb of food per day (according to the USDA), or 2.13kg, to get 1mM (molality technically when dealing with dry weight, or mols per kg) of MB in the food would require consuming ~681mg. This is still lower than what the flies actually consumed, since a human eats less food per mass than a fly, and will have a much lower MB serum level even at this consumption. If you wanted 1mM in the blood, you'd need to take around 1.6 grams of MB, which is in line with the calculated LD 50 for MB.

MB only shows any signs of toxicity at calculated levels above 600mg in humans of standard weight (or about 10mg/kg - this is based on the rat data), and will get even more toxic as those levels rise. However, we're talking about a daily consumption of 1mg (or 0.01-0.02mg/kg!). That's, again, 200 to 1000 times LOWER than used in medicine over all these years. We've seen that any life enhancing effects, and even the brain and memory and learning enhancing effects, have to do with MB being in the correct proportion to cytochrome c. It's a narrow range between 10-1000nM, seeming to peak at 100nM (1mg for a standard person of my weight), non-existent at 10nM, and over halved by 1000nM. I highly doubt, with such minute amounts, that one has to worry about any of these dilemmas you are throwing out, since we are orders of magnitude below levels that have never been documented to cause them in the first place.

Common sense kinda prevails here. But if you can cough up the proof that can counter my proof (such as that MB is an antioxidant, not a ROS generator, in the nanomolar ranges outside of being properly energized by supra sunlight level red light) then of course I'll listen to it. But so far you've done no such thing.

Edited by geddarkstorm, 28 February 2009 - 12:28 AM.

[geddarkstorm]

I need to make a correction about lipofuscin. It can generate singlet oxygen like MB (this isn't the same as super oxide either) at a blue-light wavelength of 420-430nm. Nevertheless, this doesn't appear to be the only way lipofuscin causes damage, and may well simply be a product of the products of lipofuscin A2E oxidation (and lipofuscin also kills by stopping retinal cycling and directly stimulating the apoptosis cascade, irregardless of singlet oxygen). The reactive aldehydes and ketones generated by lipofuscin are more dangerous than singlet oxygen it would appear. Singlet oxygen is also generated by retinal itself, and is something your eye always has to be equipped to deal with throughout life.

But you are right on that point, Krillin, that lipofuscin can generate singlet oxygen (after what threshold amount of light energy, I don't know) like MB, though MB is an exogenous factor not a natural byproduct. And since over half of macular degeneration cases, at least, are linked to specific genes and not directly from photosensitization to make singlet oxygen, my statement stands just the same. Our retinas are definitely not prone to sensitization damage (nevermind that sunlight lacks the energy to start sensitization by MB), and are designed to deal with singlet oxygen. You live your whole life, or up to the very end, without experiencing it (the large majority never experience it), as macular degeneration mostly only happens much later in life once cells start tiring out and dying throughout your entire body, and more specifically if you have the wrong combination of genes.

In fact, one could predict MB would protect from macular degeneration by delaying cellular senescence, and attenuating super oxide and other forms of more damaging types of ROS. Probably why it isn't a surprise that direct injection into rabbit eyes does not impair retina function.

Edited by geddarkstorm, 28 February 2009 - 06:36 PM.

[automita]

it turns skin blue

[geddarkstorm]

it turns skin blue

At high doses, at least above 200mg, and most probably around the really high doses for malaria treatment, not less than. 1mg won't change the shade of your skin any, but it might slightly tint your urine in the morning

Now if you bathe in the stuff... I hear that's the magical elixir for smurf transformation.

Edited by geddarkstorm, 01 March 2009 - 05:10 PM.

[krillin]

I knew the laser was way more powerful than sunlight, and I never said that retinas have no capacity to heal. Why do you think I wrote "Damage would probably be very slow to show up"? The indisputable facts are that

1. MB can generate ROS at nanomolar concentrations.

When hit with a powerful enough, proper wave length, light source - but not before then.

Where's the reference for this? You only provided a reference for a concentration threshold, and it said "A decrease of the fluence rate from 100 to 50 mW/cm2 increased the phototoxic response"

2. MB can generate ROS at solar intensities.

No. You have shown no proof of that, at all. I have seen no proof of that, at all.

Reread my posts. Search them for the phrase "solar simulator". I also gave you the google search words to find MB + sunlight water disinfection processes.

Common problem? Age related macular degeneration is the highest cause leading to legal blindness with age, yes. However, it's estimated that only 10 percent of the population gets it over the age of 55, and up to 30 percent over the age of 75. The large majority of people do not get it. It would depend on what you classify as common if this is common or not. Furthermore, it is genetically caused predominately, not related to oxidative stress (non-carriers of the risk gene only have a 22% chance of macular degeneration by age 95). So no, our antioxidant systems are quite good enough for the vast majority of people.

A 22% risk is large to me. Would you casually dismiss a 22% unemployment rate too?

I believe that if their rates went up significantly, someone would have noticed that after all this time - as we've had plenty of time to notice such changed rates. Perhaps I just have more faith in medical and scientific observations. They got us this far, so far, after all.

We're far closer to ignorance than you believe. It wasn't until 2008 that someone finally published a U-curve for vitamin D and mortality. (PMID: 18695076 The full text says 40-49 ng/ml is best, and mortality gets worse above 50 ng/ml.) People like you scoffed at my demanding caution with vitamin D, but I was vindicated in short order.

I apologize for making you type a response to the fly study. I thought that by saying that it doesn't count I was clearly communicating that I was not using it to help make my case.

Here are some new reasons to worry about MB. PMID: 9714855 says at 50 nM, MB antagonizes nitric oxide. (Abstract says 1-100 nM, text shows a figure with 50 nM.) My quick study of the subject indicates that (eg. PMID: 8095358) the mechanism is generation of ROS that prevent NO from activating guanylate cyclase to create cGMP.

PMID: 7858245 says one mechanism by which estrogen increases bone mass is through the cGMP pathway that is inhibited by MB. Other papers say that bone blood flow is involved, and that MB also interferes with NO's antithrombotic effect. See PMID: 9728516, 8711376, 8624735, 8093254.

And finally, MB's irreversible inhibition of prostacyclin synthesis through a non-cGMP mechanism. Suppression of prostacyclin is probably how Vioxx killed people. 100 nM is at the extreme low end of the studied range, so it's uncertain if your protocol will affect this significantly.

Br J Pharmacol. 1989 May;97(1):51-6.
Methylene blue but not changes in cyclic GMP inhibits resting and bradykinin-stimulated production of prostacyclin by pig aortic endothelial cells.
Martin W, Drazan KM, Newby AC.
Department of Pharmacology, University of Glasgow.

1. Primary cultures of pig aortic endothelial cells produced 6-keto-prostaglandin F1 alpha (6-keto PGF1 alpha), the stable breakdown product of prostacyclin, both in the resting state and in response to bradykinin. The rise in 6-keto-PGF1 alpha production induced by bradykinin (1-100 nM) was concentration-dependent. 2. Treating endothelial cells with the inhibitor of soluble guanylate cyclase, methylene blue (0.1-20 microM) produced an irreversible reduction in resting and bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha with an IC50 of 0.5 +/- 0.1 microM. Treating endothelial cells with haemoglobin (10 microM) had no effect on resting or bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha. 3. Two stimuli that elevate the level of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in endothelial cells, 8-bromo cyclic GMP (30 microM) and atriopeptin II (0.1 microM), each had no effect on resting or bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha. Furthermore, treating endothelial cells with either 8-bromo cyclic GMP (30 microM) or atriopeptin II (0.1 microM) had no effect on the ability of methylene blue (20 microM) to inhibit resting or bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha. 4. Adding arachidonic acid (1 microM) to endothelial cells led to a marked stimulation of 6-keto-PGF1 alpha production. Treating cells with either methylene blue (20 microM) or the cyclo-oxygenase inhibitor, flurbiprofen (10 microM), inhibited both resting and arachidonic acid (1 microM)-induced production of 6-keto-PGF1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 2541859

[kismet]

Common problem? Age related macular degeneration is the highest cause leading to legal blindness with age, yes. However, it's estimated that only 10 percent of the population gets it over the age of 55, and up to 30 percent over the age of 75. The large majority of people do not get it. It would depend on what you classify as common if this is common or not. Furthermore, it is genetically caused predominately, not related to oxidative stress (non-carriers of the risk gene only have a 22% chance of macular degeneration by age 95). So no, our antioxidant systems are quite good enough for the vast majority of people.

A 22% risk is large to me. Would you casually dismiss a 22% unemployment rate too?

I don't want to comment on anything else, but double digit numbers are actually huge incidence rates (=very common) in terms of epidemiology. I really wouldn't dismiss it as anything short of what it is. A disease which is going to affect tens of millions of people per year (worldwide) is quite common to me.
Those diseases which are even more wide-spread (e.g. vacsular calcification) sometimes are described as "universal" (to contrast it with diseases which are just "very common").

[geddarkstorm]

Where's the reference for this? You only provided a reference for a concentration threshold, and it said "A decrease of the fluence rate from 100 to 50 mW/cm2 increased the phototoxic response"

You are absolutely correct. It was a critical MB concentration, rather than light, that they saw in their study as the determinant for singlet oxygen production. It is indeed a complete error on my part that mixed the two up.

On the other hand, numerous studies have shown MB to act as a wonderful antioxidant. So it falls into that same paradigm of too much antioxidant becomes pro-oxidant, which is dependent on the chemical in question of course.

Reread my posts. Search them for the phrase "solar simulator". I also gave you the google search words to find MB + sunlight water disinfection processes.

I stand corrected here, then. Which brings us right back around to concentration.

None the less, its use medicinally in humans has not shown these sensitization effects or production of ROS except at the very high doses such as used in malaria treatment, and even then this is more specific for the malaria parasite's reductases, and doesn't happen in human cells to the same extent (i.e. doesn't damage them oxidatively like it does the parasite). Non laser light, or sunlight exposure doesn't change that significantly apparently, or it would be used (or would have to be avoided). Add in MBs antioxidant protection of cells and neurons in animals and in vivo, except when hit with a powerful enough laser as in photodynamic therapy, when at high enough concentrations (and even then, collateral (non-tumor) damage by MB is apparently very low).

A 22% risk is large to me. Would you casually dismiss a 22% unemployment rate too?

I think comparing it to unemployment is a bit more than misleading, and you aren't suddenly blind the moment you get macular degeneration, as that 22% is accounting for any level of it. Notice the age ranges it occurs in too. It isn't something that just happens to people in general. Like most degenerative diseases, it's linked to some loss of function in repair or normal cellular processes (senescence begins), or chronic inflammation (why most macular degeneration is linked to complement system genes).

It raises progressively to 22% if someone lives long enough to 95, as does the percentage occurrence for a huge suite of degenerative events. As Kismet points out, that would be considered quite common on the medical side of things, but it is nevertheless not the majority. Now, if cellular senescence is staved off, healing is allowed to occur, then it is likely to be averted or resisted for a longer period of time. MB does give that potential. ROS itself is many times more a symptom of an ailing cell than a cause, on the molecular level, which is a reason antioxidants don't expand lifespan significantly or consistently on their own, so treating one of the sources, mitochondrial dysfunction, is therein highly advantageous in our current view and knowledge.

We're far closer to ignorance than you believe. It wasn't until 2008 that someone finally published a U-curve for vitamin D and mortality. (PMID: 18695076 The full text says 40-49 ng/ml is best, and mortality gets worse above 50 ng/ml.) People like you scoffed at my demanding caution with vitamin D, but I was vindicated in short order.

I never scoffed about vitamin D caution, and I don't just mean here, but have warned about too much vitamins on these boards before. Although I can't see that paper at the moment and look at how mortality drifted above 40-49ng/ml (which is a lot of vitamin D already, if 17ng/ml is the cut off point before deficiency on the low end), so it may not be a significant change compared to the low end, it still goes to show that there's an optimal for all things. My general stance is that if a compound is involved directly as cofactor, in metabolism, or as a hormone, then it likely shouldn't be messed with too much if it isn't already out of whack/deficient, though everything should be investigated and with caution and skepticism anyways. Nevermind that vitamin D is produced by our own bodies and not an actual vitamin. Though, we still probably don't get enough sunlight to produce the proper amounts in the modern era, and supplementation at some rate is probably useful and recommended. We are designed to spend our lives in the sun, not cooped in houses and offices under a tenth the lighting.

I apologize for making you type a response to the fly study. I thought that by saying that it doesn't count I was clearly communicating that I was not using it to help make my case.

Well then, why bring it up at all : p? But, nah, don't apologize. It's an interesting study, and once more hits home the important lesson that there are ranges for chemicals to be useful, and beyond which they lose their particular use or become very dangerous.

Here are some new reasons to worry about MB. PMID: 9714855 says at 50 nM, MB antagonizes nitric oxide. (Abstract says 1-100 nM, text shows a figure with 50 nM.) My quick study of the subject indicates that (eg. PMID: 8095358) the mechanism is generation of ROS that prevent NO from activating guanylate cyclase to create cGMP.

PMID: 7858245 says one mechanism by which estrogen increases bone mass is through the cGMP pathway that is inhibited by MB. Other papers say that bone blood flow is involved, and that MB also interferes with NO's antithrombotic effect. See PMID: 9728516, 8711376, 8624735, 8093254.

And finally, MB's irreversible inhibition of prostacyclin synthesis through a non-cGMP mechanism. Suppression of prostacyclin is probably how Vioxx killed people. 100 nM is at the extreme low end of the studied range, so it's uncertain if your protocol will affect this significantly.

Br J Pharmacol. 1989 May;97(1):51-6.
Methylene blue but not changes in cyclic GMP inhibits resting and bradykinin-stimulated production of prostacyclin by pig aortic endothelial cells.
Martin W, Drazan KM, Newby AC.
Department of Pharmacology, University of Glasgow.

1. Primary cultures of pig aortic endothelial cells produced 6-keto-prostaglandin F1 alpha (6-keto PGF1 alpha), the stable breakdown product of prostacyclin, both in the resting state and in response to bradykinin. The rise in 6-keto-PGF1 alpha production induced by bradykinin (1-100 nM) was concentration-dependent. 2. Treating endothelial cells with the inhibitor of soluble guanylate cyclase, methylene blue (0.1-20 microM) produced an irreversible reduction in resting and bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha with an IC50 of 0.5 +/- 0.1 microM. Treating endothelial cells with haemoglobin (10 microM) had no effect on resting or bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha. 3. Two stimuli that elevate the level of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in endothelial cells, 8-bromo cyclic GMP (30 microM) and atriopeptin II (0.1 microM), each had no effect on resting or bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha. Furthermore, treating endothelial cells with either 8-bromo cyclic GMP (30 microM) or atriopeptin II (0.1 microM) had no effect on the ability of methylene blue (20 microM) to inhibit resting or bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha. 4. Adding arachidonic acid (1 microM) to endothelial cells led to a marked stimulation of 6-keto-PGF1 alpha production. Treating cells with either methylene blue (20 microM) or the cyclo-oxygenase inhibitor, flurbiprofen (10 microM), inhibited both resting and arachidonic acid (1 microM)-induced production of 6-keto-PGF1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 2541859

Those are all very good and valid criticisms and safety concerns with MB. As with all things, there's a risk/benefits scale to evaluate, some things are intrinsically much more tipped to one end or the other. Could these really low doses of MB affect hypertension and cardiovasculatory health in a negative way? Perhaps. Could these really low doses increase cellular health and all body functions in a positive way? Perhaps. Which one will win out in vivo? The rat toxicology studies didn't see any problems with anything even remotely near the level of MB I'm talking about in vivo. This might well represent an order of magnitude difference caused by the in vivo environment over the in vitro environment where many of our studies come from. Since the clinical affects of MB in humans, and the toxicological effects in rats are very well documented, I have to say the benefits far exceed the risks in my opinion in this matter, mostly because the level of MB being used is vanishingly small compared to where it has been demonstrated to cause any problems in vivo, and where it is actually used medically for decades and decades. Additionally, some lifespan increases in rats have even been seen. Plus, you've got that cool significant increase in learning and memory in rats, related to the exact same processes that cause reversal of mitochondrial/cellular senescence by MB. Will it happen in humans? Well, no one has tried to use so little MB for anything in a person, so we'll have to see!

MB has been extensively looked at medically, it isn't one of those "we are probably ignorant" sorts of things, I think. Vioxx is a perfect example where a problem was rapidly seen (on the grand scale of things), and dealt with. If the same happened with MB, we'd already know, as it's been looked at for a plethora of medical uses, and characterized over so many years at so many levels. New research could always change our views on things, always, but for now this is my stance. If one worries about these effects by MB, just take their counter, like resveratrol and/or quercetin, which counters all these potential adverse effects, including photosensitization (by being antioxidants), NO inhibition (by stimulating the pathways directly in some circumstances/cells, like the vascular tissue and neurons), and potential platelet aggregation from low PGF1-alpha (by acting to directly inhibit platelet aggregation, though MB actually quite weakly acts directly as a clot inhibitor too); all while at the same time giving their own suite of benefits. So, everything comes into balance.

Edited by geddarkstorm, 04 March 2009 - 08:46 AM.

[geddarkstorm]

There are two final points I'd like to make real fast.

Firstly, after 2 months of 1mg MB (with 5 consecutive days a week, 2 days off sort of cycling), my blood pressure was 100/60 while being quite nervous and tense during my first ever attempt to donate blood -- same level its been most of my life.

Lastly, while you are completely right that 100micromolar through millimolar amounts of MB can kill a high percentage of microbes in water sitting in a clear jug out in the sun for a few hours, why is it that photodynamic therapy (PDT) uses red light diodes with energies from 565mW/cm^2 (also notice no collateral damage) to 100W? That's nearly 5 to 1000 times the energies of the entire solar spectrum, let alone the actual solar energy just of the red light spectrum MB absorbs. It'd be more cost effective if one could use lower powered diodes, but there's a reason such high power plus high micromols of MB have to be used. Maybe in vivo just isn't quite the same as a jug of water; or there's a fundamental sensitivity difference between microbials and our cells.

Edited by geddarkstorm, 04 March 2009 - 04:34 PM.

[guaif1]

http://www.fasebj.or...stract/22/3/703

Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways
Hani Atamna¹, Andy Nguyen, Carla Schultz, Kathleen Boyle, Justin Newberry, Hiroyuki Kato and Bruce N. Ames Nutrition and Metabolism Center, Children's Hospital Oakland Research Institute, Oakland, California, USA





¹Correspondence: Nutrition & Metabolism Center, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA, 94609-1673, USA. E-mail: hatamna@chori.org<script type="text/javascript">

Methylene blue (MB) has been used clinically for about a century to treat numerous ailments. We show that MB and other diaminophenothiazines extend the life span of human IMR90 fibroblasts in tissue culture by >20 population doubling (PDLs). MB delays senescence at nM levels in IMR90 by enhancing mitochondrial function. MB increases mitochondrial complex IV by 30%, enhances cellular oxygen consumption by 37–70%, increases heme synthesis, and reverses premature senescence caused by H₂O₂ or cadmium. MB also induces phase-2 antioxidant enzymes in hepG2 cells. Flavin-dependent enzymes are known to use NAD(P)H to reduce MB to leucomethylene blue (MBH₂), whereas cytochrome c reoxidizes MBH₂ to MB. Experiments on lysates from rat liver mitochondria suggest the ratio MB/cytochrome c is important for the protective actions of MB. We propose that the cellular senescence delay caused by MB is due to cycling between MB and MBH₂ in mitochondria, which may partly explain the increase in specific mitochondrial activities. Cycling of MB between oxidized and reduced forms may block oxidant production by mitochondria. Mitochondrial dysfunction and oxidative stress are thought to be key aberrations that lead to cellular senescence and aging. MB may be useful to delay mitochondrial dysfunction with aging and the decrease in complex IV in Alzheimer disease.—Atamna, H., Nguyen, A., Schultz, C., Boyle, K., Newberry, J., Kato, H., Ames, B. N. Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways.

So what do you think about that?

[krillin]

Now, if cellular senescence is staved off, healing is allowed to occur, then it is likely to be averted or resisted for a longer period of time. MB does give that potential. ROS itself is many times more a symptom of an ailing cell than a cause, on the molecular level, which is a reason antioxidants don't expand lifespan significantly or consistently on their own, so treating one of the sources, mitochondrial dysfunction, is therein highly advantageous in our current view and knowledge.

Neurons are postmitotic, so can they even go senescent? Senescence doesn't seem to me to be a very useful thing to target. From p. 233 of Ending Aging

The use of the word "senescent" to describe these cells is, however, a bit misleading. When people hear about these cells, they often assume that cellular "senescence" is the ultimate fate of all of the cells in the body with age, and that the entry of "young" cells into this senescent state is the underlying cause of aging. [. . .] In fact, senescent cells are generally held to be extremely rare even in very aged people.

His solution is to just target senescent cells for elimination. But if you want to slow the process down, alternatives to MB include resveratrol, carnosine, MitoQ (And maybe ubiquinol too. It did help those SAMP mice.), Alcar/RALA, and oleuropein. Most of that list also treats mitochondrial dysfunction, so I wouldn't expect MB to provide much in the way of an additional benefit to the heavier supplement stacks.

We're far closer to ignorance than you believe. It wasn't until 2008 that someone finally published a U-curve for vitamin D and mortality. (PMID: 18695076 The full text says 40-49 ng/ml is best, and mortality gets worse above 50 ng/ml.) People like you scoffed at my demanding caution with vitamin D, but I was vindicated in short order.

I never scoffed about vitamin D caution, and I don't just mean here, but have warned about too much vitamins on these boards before. Although I can't see that paper at the moment and look at how mortality drifted above 40-49ng/ml

I didn't intend to characterize you as a vitamin D caution scoffer. I was just trying to draw an analogy to other hazards that have taken decades to come to light. I'll post the D vs mortality graph in the D thread.

MB has been extensively looked at medically, it isn't one of those "we are probably ignorant" sorts of things, I think. Vioxx is a perfect example where a problem was rapidly seen (on the grand scale of things), and dealt with.

That was an acute effect, and I believe that subtle chronic effects are much harder to discover.