Dynamised Preparations in Cell Culture?

March 15, 2010

Effects of Homeopathy in cell culture: O rly?


This post concerns this paper : “Dynamized Preparations in Cell Culture” — Sunila et al. 6 (2): 257 — Evidence-based Complementary and Alternative Medicine

I discovered this paper whilst taking a look at this website which is well worth a read, if ever feel you might want to learn about telepathic DNA (no. srsly) or absorb life threatening amounts of stupid.

Sunila et al want to look at the effect of homeopathic remedies on cells in a cell-culture setup. They also want to use homeopathic remedies rumoured to be effective in combating cancer. The hypothesis would seem to be that if the cells treated with homeopathic remedies die in cell culture then OMG! Homeopathy will cure cancer!

They use a set of homeopathic remedies, and compare mother tinctures (MTs), 30C preparations and 200C preparations.

So they take some cells, and add some homeopathic remedies to them. They then assay for stuff like cell viability (via MTT or tryptan blue exclusion method ) cell proliferation via tritiated thymidine uptake assay, and CHO cell colony formation, p53 message upregulation via RT-PCR and DNA damage via DNA laddering assay. Fine.

One of the first things that annoys the pants off me about this paper is that they seem to chop and change cells types for each different set of assays, seemingly arbitrarily. Why this is, I don’t know. If I was feeling uncharitable, I might say that this is because assay results they see only occur for certain cell types. If.

What they should have done (IMHO) is either do some experiments for all types of cells, or just use one cell type for all experiments. Chopping and changing like this makes comparison between the different assays dubious at best.

If you can’t get at the paper, or don’t want to plough through it, here are the results in bullet point form:

  • Tryptan blue exclusion method: Dalton’s lymphoma ascites &  Ehrlich ascites carcinoma cells – (figures 1 & 2 in the paper).

    • “Results on DLA cells and EAC cells were almost similar.”

      • Yes – “Almost similar” – I’ve never seen that phrase in a paper before, either.
    • Negative controls: Cells live.
    • Postive controls: There are no positive controls.
    • 200C Conium, Carcosinium,  MTs of Condurango, Hydrastis & Phytolacca and 200C preps of Thuja  & Hydratis: Some cells Die.
    • Thuja & Lycopodium mother tintcures (contain active ingredient). ~All cells die.
  • Long term viability of L929 cells is quite drastically reduced in most MTs, and to a lesser extent in some of the 30C and 200C preps (fig3).
  • They show that pretty much all their remedies, but not the controls reduce CHO cell colony formation (fig4).
  • They show that their MTs significantly reduce tritiated thymidine uptake (fig5).
  • They show that Thuja seems to induce DNA damage (fig6) and that Carcosinium seems to induce p53 expression (fig7).
  • Table 1 shows that all but one of their MTs induces apoptosis, and fewer 30C and fewer again 200C preps cause apoptosis, as determined (subjectively) by morphology.

The dye exclusion method, CHO clustering & visual assessment of apoptotic phenotype are all subjective “looking down a microscope” assays, and *may* be subject to bias. Just saying, is all.

So, we’ve got a fair old batch of results to analyse. The assays are pretty standard, and so assuming they were done competently, methodology appears fine – although there is a caveat wrt the dye exclusion assays – which measure cell viability indirectly by measuring cell membrane integrity – but probably not an issue here.

However, they do not state how many times the experiments were repeated – n=2? n=3? n=400? who knows?

There is one teensy weensy little caveat in the paper, mentioned in the methods, tucked under the table of remedies used:

“Ethanol content in the preparations may vary and no attempt was made to determine the exact content. Maximum ethanol concentration used in the experiment was 2%, which will not produce any effect on cells. Moreover results were compared with dynamized vehicle control or non-dynamized alcohol control.”

The alcohol control was only used in one assay- the p53 RT-PCR assay. All the others were controlled with “dynamized vehicle control” – which sounds like some kind of groovy new steering feature on a formula 1 car – but is, as far as I can see, just shaken phosphate-buffered saline. Which won’t contain ethanol.

But hey! Remeber, that doesn’t matter, because “Maximum ethanol concentration used in the experiment was 2%, which will not produce any effect on cells.

But they don’t provide a reference for this. This would appear to be an assumption made, and not supported, or checked, or referenced in any way.

To be fair, 2% Ethanol, doesn’t sound like much -  however, a bit of high school chemistry can show you that 2% Ethanol solution corresponds to 435mM Ethanol.

Mwt of ethanol ~ 46g
100% (pure) ethanol = 1000/46 = 21.7M
2% Ethanol = 21.7*0.02 = 0.435M Ethanol.

Which is actually a pretty high concentration. Even if the remedies only contain 1% ethanol, we’d still have 4.35mM ethanol in our cell culture, which is more than enough of a compound to have an appreciable effect in cell culture.

But we don’t know what alcohol is in the remedies – The authors didn’t check. “no attempt was made to determine the exact content” – this is really bad science. They didn’t even bother to check.

I thought it would be fun to go to the Willmar Schwabe (suppliers of the remedies) website, and see if there is any information regarding alcohol content of their remedies.

This page reveals that their mother tinctures are “The therapeutically active ingredients in many cases are extracted in alcohol-water mixture of varying percentages of alcohol depending on the solubility, stability and extractability of the potential ingredients. Such alcoholic extracts are called “Mother Tinctures”.”

So the MTs will definately contain some ethanol.
What about the 30C and 200C remedies?

Well, according to this:
“The use of genuine raw materials, back potency and expensive and purest form of alcohol, namely Extra Neutral Alcohol (ENA) make Schwabe India dilutions superior to other dilutions available in the market. Extra Neutral Alcohol guarantees that the dilutions and mother tinctures are free from impurities.”

The 30C and 200C remedies will also definately contain some ethanol.

So, to sumarise:

  1. The MTs contain planet extract in an alcohol/water mix, of unknown proportions and composition.
  2. The 30C and 200C preps contain an unknown alcohol/water mix (+ water memory of original compounds, OBVIOUSLY)

I’ve e-mailed Willmar Schwabe asking for more specific information about the composition of these remedies… as and when I get a response, I’ll update this blog post.

Given that we have established that there is most likely *some* ethanol in all these remedies, and paying particular attention to the assays caried out in Sunila et al, just what are the effects of low concentrations of Ethanol in cell culture?

1) Effects of alcohol on cell viability.

Ethanol concentrations as low as 10mM lead to increased necrosis and apoptosis (i.e. cell death), and DNA fragmentation, another assay used in Sunila et al.

Castilla, R et al, DUAL EFFECT OF ETHANOL ON CELL DEATH IN PRIMARY CULTURE OF HUMAN AND RAT HEPATOCYTES (2004) Alcohol & Alcoholism Vol. 39, No. 4, pp. 290–296, 2004 doi:10.1093/alcalc/agh065,

2) Effects of alcohol on cell clustering

“Low concentrations of ethanol (5-10 mm) inhibited cell-cell adhesion
An appreciable decrease in cell clustering is observed in low concentrations of alcohol – one of the measures of the effect of homeopathic remedies used in this paper.

Charness, ME et al. Ethanol Inhibits Neural Cell-Cell Adhesion, (1994), J. Biol. Chem, Vol. 269, No. 12, Issue of March 25, pp. 9304-9309

3) Effects of alcohol on thymidine uptake.

Suppressed levels of Thmidine uptake in the cells exposed to the MTs and remedies might be due to the fact that “alcohol levels greater than or equal to 50 mg/dL suppressed tritiated thymidine uptake of normal lymphocytes”

Glassman AB et al. Effects of ethyl alcohol on human peripheral lymphocytes. (1985) Arch Pathol Lab Med. Jun;109(6):540-2.

4) Effects of alcohol on p53 expression

Ethanol causes Oxidative stress – Sunila et al assayed for p53 expression – p53 expression known to be induced by oxidative stress.

Han, E.S. et al The in vivo gene expression signature of oxidative stress. (2008) Physiol Genomics. 12;34(1):112-26.

5) Effects of Alcohol on DNA fragmenting:

Castilla, R et al, again.

This paper shows that 10mM concentrations of Ethanol lead to increased necrosis and apoptosis, and DNA fragmentation, another assay used in Sunila et al.


Now. Given that there is published, peer-reviewed evidence that suggests that all the effects observed in Sunila et al can be explained by the presence of small amounts of ethanol, and that, the manufacturers state that all the remedies used in this Sunila et al will contain small amounts of ethanol, is it not more plausible and rational to conclude that the effects observed are caused by the small amounts of ethanol present?

Differences between different preps might be explained by different ethanol/water ratios. Differences between the effect of different MTs and MTs and 30C/200C preps can be explained by the presence in the MTs of actual, measurable amounts of chemicals that may have an effect upon cells in culture.

All this uncertainty could have been solved with a bit of Infrared spectroscopy, and better design of controls, to contain an appropriate amount ethanol.

However, such controls might well have just shown that these in vitro effects of homeopathic remedies are all down to the alcohol content. Just like Frenkel et al. (see Dr Rachie’s post and Orac’s post).

It would seem that when it comes to cell culture, homeopaths have some sort of cognitive blind spot which prevents them providing suitable controls for the solvents in which their homeopathic remedies are… remembered? One would hope that given the homeopathic preocupation with the memory of solvents, that they might remember to use the correct solvents in their controls.


Antibiotics: take the full course!

February 14, 2010

Good science, for a change. Not woo.


In this season of ear, throat and chest infections, there is a good chance that you, or someone you know, will have been prescribed antibiotics recently.

When given antibiotics, your doctor, and your pharamcist will/should remind you to take the full course of antibiotics.

This paper just out in Molecular Cell explains why.

If you fail to take the full dose of antibioitics, or you miss a dose, you are potentially exposing the bacteria to a sub-lethal dose of antibiotic.

According to this work from Boston & Harvard Universities, sub-lethal doses of a variety of antibiotics (including Ampicilin, a popular choice for GPs to prescribe), rather than killing the bacteria, cause a stress response in the bacteria, which in turns leads to prodution of reactive oxygen species (ROS). Kohanski et al showed that this increase in ROS production can cause up to an 8-fold increase in the mutation rate in E.coli. They confirmed that ROS was the cause by treating the bacterial simultaneously with sub-lethal doses of antibiotics, and thiourea (which limits ROS production). The thiourea returned mutation rate nearly to control levels.

ROS can directly cause random damage to the bacterial genome, leading to an accumulation of mutations. ROS can also lead to the activation of SOS genes, which repair DNA – however, in doing so, they can also introduce mutations.

Taken from here. No permision given, but fair use claimed.

Some of these mutations may confer antibiotic resistance upon a bacteria. Which means your bugs may now survive the course of antibiotics.

The upshot of which is you need more antibiotics, and you may have created your very own drug resistant form of a pathogenic strain of bacteria (think MRSA).

WooHoo! Go YOU!

 

 


Homeopathy and Anaesthesia.

February 6, 2010

Addressing the oft-repeated mantra – “you don’t know how anaesthetic works”


When considering homeopathy, an obvious bone of contention is the lack of a plausible molecular mechanism .

When proffering this as a reason why homeopathy is not feasible, a homeopath will typically counter with “well what about anaesthesia – we don’t know how that works either.”

So lets explore this a little further…

“General anaesthesia is administered each day to thousands of patients worldwide. Although more than 160 years have passed since the first successful public demonstration of anaesthesia, a detailed understanding of the anaesthetic mechanism of action of these drugs is still lacking.” So states the opening gambit of an excellent and fairly recent review of the potential receptors for general anaesthetics.

Kopp Lugli, A., Yost, C.S. and Kindler, C.H. (2009) “Anaesthetic mechanisms: update on the challenge of unravelling the mystery of anaesthesia”. European Journal of Anaesthesiology, 26(10), p 807–820

A general anaesthetic has to do three things: cause immobility, amnesia and unconsciousness. Many potential molecular targets are being researched – these are summarised in the table below.

Taken directly from Kopp Lugli, A., Yost, C.S. and Kindler, C.H. (2009) “Anaesthetic mechanisms: update on the challenge of unravelling the mystery of anaesthesia”. European Journal of Anaesthesiology, 26(10), p 807–820. Fair use claimed.

We don’t know for sure what all the molecular targets are, but Franks, N.P. (2006) “Molecular targets underlying general anaesthesia” Br J Pharmacol. 147(S1): S72–S81, is a decent review of what is known about the molecular mechanism of two types of Anaesthetic, propofol and etomidate, and their interaction with the GABA-A receptor. The GABA-A receptor is a ligand-gated ion-channel, a complex of proteins that allows ions (in this case Chloride ions) to cross an otherwise impermeable membrane. GABA-A receptors are found in most areas of the brain. Propofol and etomidate are positive allosteric modulators of the ion channel – they bind at a site on the ion-channel distinct from the pore through which the ions pass and make the ion-channel more easily openable, and Cl- ions can flow freely accross the membrane. This will lead to hyperpolarization of the neurons, which in turn, inhibits neurotransmission, which eventually leads to the anaesthetic effect.

Science doesn’t know the precise ordering of molecular events that leads to sucesful anaestheisia – but it is working on it – and it is making progress. But for all anaesthetics we have “leads” – potential, rational, explainable targets for their mode of action.

So we actually know a lot more about the mechanism of action of anaesthetics, than we do about homeopathy.

Now lets look at the formulation of a typical general anaesthetic.

Propofol is distributed as Diprivan – a 1% solution in combination with various inert stabilisers and anti-microbial agents.

The dosing guidlines for Diprivan states: “adult patients under 55 years of age and classified as ASA-PS I or II require 2 to 2.5 mg/kg of DIPRIVAN Injectable Emulsion for induction when unpremedicated or when premedicated with oral benzodiazepines or intramuscular opioids. For induction, DIPRIVAN Injectable Emulsion should be titrated (approximately 40 mg every 10 seconds) against the response of the patient until the clinical signs show the onset of anesthesia. “

Lets assume we have a 100kg adult, and we are going to dose them at 2.5 mg/kg.

So that’s a total of 2.5mg x 100 = 250mgs of Diprivan.

The molecular weight of propofol is 178.271 g/mol.
This is 0.25g / 178.271 = 0.00140235 moles of Diprivan.

Multiply this by Avagadro’s Constant and you discover that this 250mgs has got 8.44×10^20 molecules of pharmacologically active ingredient.
844,000,000,000,000,000,000 molecules.

Contrast this with a Homeopathic anaesthetic, which would have zero molecules of pharmacologically active ingredient.

Interestingly enough, given all the things homeopathy claims to cure (cancer, AIDS, Homosexuality, etc) – a cursory piece of googlage actually reveals that one of the few things homeopathy doesn’t claim to do is general anaesthetic – fear of pain is a great leveller, I guess.

(If you can find a reference to a Homeopathic Anaesthetic – please leave a comment below!)

However, homeopaths naturally claim that homeopathy can help with after effects of anaesthesia and surgery – after effects which coincidentally, will resolve naturally more often than not.

This reference sheet entitled “Homeopathic support during surgery” is particularly shocking.

The first thing to note is that it states: “Use 30c potency for these acute treatments.”
As previously established – 30c potency contains zero pharmacologically active molecules.

Of all the treatments outlined by this sheet – the most dangerous and irresponsible are those that detail treatment for symptoms of surgical site infection (SSI).

“Hepar Sulph wounds that have become weeping and pustulent”
“Pryogen indicated when there is necrosis, gangrene or dead flesh, in the wound”

That is exceptionally irresponsible – Between 5% and 10% of people who undergo an operation suffer from an SSI. Dangerous pathogenic bacteria such as MRSA and Clostridium difficile can thrive in the wounds left after surgery, and if left unchecked, infection can be fatal. The correct treatment for an SSI is antibiotics.

If anyone were to rely on homeopathy rather than conventional antibiotics, they would be putting their lives in jeopardy.


The Molecular Mechanisms of Homeopathy.

January 10, 2010

With the launch of the 1023 campaign against homeopathy, it seems appropriate for me to write a bit on that very subject. But where to start – there are so many blogs that talk about the issues that people have with homeopathy.  The ethical reasons for campaigning against it and the slap in the face that homeopathic “medicine” is to scientific thinking have been subject to in-depth coverage. Recently Prof David Colqhoun has finally gained access to teaching materials for a now-defunct BSc in Homeopathy, via a freedom of information request. He is reviewing them systematically on his excellent DC’s improbable science blog.

So with all this covered by other, more eloquent and seasoned bloggers than myself, I have decided to look at homeopathy from my own niche in science – from a structural/molecular biologist’s point of view.

How medicines work.

To understand the issues that science has with homeopathic “remedies”, one needs to know how conventional drugs work. They work by interacting with another molecule in your body. Generally they will either activate or inhibit the activity of protein molecule that is involved in some sort signal transduction pathway, or in the catalysis of a particular chemical reaction. Structural biology, particularly X-ray crystallography is an immensely powerful tool for understanding the nature of these drug: receptor interactions.  For example, crystallography has revealed the nature of interactions between a bacterial ribosome and the antibiotic chlorampenicol and HINI (Swine flu) neuraminidase interaction with anti-influenza drug, Tamiflu. These structures reveal the nature of the interaction at an atomic level. We can see Electrostatic interactions (negatively charged atoms/groups interacting with positively charged atoms/groups) such as salt bridges and hydrogen bonds. We can see aromatic stacking interactions (aromatic groups stacking alongside each other) and other hydrophobic interactions. We can precisely map out the atomic nature of an interaction.

Such information is invaluable in understanding how a drug works. We don’t have this information for every single drug that is used. But in time we will.

A “conventional” drug, eg Tamiflu, consists of a chemically defined active ingredient at a known and measurable concentration. Each adult dose of Tamiflu contains 75 mgs of Tamiflu. Given that Tamiflu has a molecular weight of 312.4 g/mol, each 75mg dose of Tamiflu contains 1.4×1020 (~140,000,000,000,000,000,000) molecules of Tamiflu (0.075/312.4 * Avogadro’s constant).

Your body takes up a known percentage of this drug – this is known as “bioavailability”. In the case of Tamiflu, the bioavailability is ~60% – so of the 1.4×1020 molecules of Tamiflu that you swallow, only 8.6×1019 get into your blood stream. These then circulate for until broken down/excreted. Tamiflu works by mimicking the natural substrate of influenza virus neuraminidase, sialic acid. By preventing influenza virus neuraminidase binding to endogenous sialic acid presented on the surface of your cells, Tamiflu prevents infection of host cells. Simple.

How Homeopathy works.

Homeopathic remedies are based upon highly dilute samples of a compound that causes a similar effect to the symptoms of a given disease (“like cures like”). For example, if disease X gives me hiccups, and plant extract Y gives me hiccups, I would take a dilute solution of plant extract Y to cure the symptoms of disease X.

We’ll overlook the fact that this is treating a symptom (by causing it?), and not addressing any underlying cause – because homeopathic remedies are “potentised” during their production. The originator of homeopathy was Samuel Hahnemann, who described a process whereby the effect of these cures could be enhanced by serially diluting them until one reached ultra low concentrations. Each dilution step would be accompanied with “succussion” of the solution – ten hard strikes against a soft, often leather-covered object. The wikipedia page on Homeopathy has a good description of this process.

A typical homeopathic remedy has a concentration of 30C, that is to say, 1 part compound in 10030 (so 1060). After Hahnemann first devised homeopathy, Avogadro’s constant (number of molecules in a mole of substance) was calculated, and shown to be roughly 6×1023 (hence 1023 campaign). So in a 1023 or 11.5C solution one might reasonably expect to find 1 molecule of compound. The odds of finding a single molecule of active compound of a 30C homeopathic remedy are very roughly 1 in 10,000,000,000,000,000,000,000,000,000,000,000,000 (10 million million million million million million).

When this was pointed out to the homeopaths (that their remedies had zero active ingredients) they happily discovered that the water in which the active ingredient was dissolved in had a memory and not only remembered the active ingredient (doubtless both its molecular structure and pharmokinetic properties), but the water could also pass this memory on to other water molecules. Cool, huh?

Note – the succussion process allows the water to remember the therapeutic ingredient, but not all the other stuff that the water has seen down the years. And given that a molecule or two of the water currently in the coffee I am drinking were likely to have at sometime been intimately associated with an Ebola virus particle or a molecule of the Ricin toxin – I sincerely hope this is the case. The molecular mechanism of succussion, and how it selects for particular solutes, is currently not understood.

But in order for a homeopathic remedy to work, not only does the water need to retain a memory of the active ingredient, it needs to actually mimic it, so it can then interact with the required target molecule to exert an effect. It has to adopt a similar/identical form.

How water interacts with other (bigger) molecules

Crystallographers can show that molecules such as proteins and DNA interact with solvent water atoms in quite specific ways. I’ve even generated a little picture to show precisely that:

The grey surface represents the molecular surface of a particular protein I’m working on. The red spheres represent ordered water molecules that interact with the suface of the protein.

We see these bound water molecules in pretty much every crystal structure we solve, as the vast majority of crystals are formed from aqueous solutions. Indeed, water molecules can become trapped inside proteins and help them fold correctly and bind substrates or metal ions. Water molecules also participate in chemical reactions – often being split to protenate leaving groups.

So the idea that water adopts a structure around another molecule is not that far fetched. So, being inquisitive, scientists tried to determine if this water structure persisted after the molecule was removed. And to their surprise, it did – but for only ~50 femtoseconds (0.00000000000005 seconds – not very long at all – and not much use for a remedy that’s going to sit on the shop shelf for weeks).

And another thing…

For the sake of further enquiry, let’s assume that the peer-reviewed letter to Nature above is wrong (it isn’t), and water can retain a memory of what’s been dissolved in it for much longer than 50 femtoseconds (it can’t).

This isn’t enough – an empty shell of water molecules around a compound that was once there isn’t going to be enough to interact with and engage protein receptors – they actually have to then rearrange and mimic the chemical and physical properties of the molecule that induced the formation of that shell in the first place.

Not only this – this water structure then has to be robust enough to occupy a protein-binding site and fend off other molecules that might normally bind to that site with a high affinity – molecules that will displace water molecules all the time in order to get into that very binding site. The original therapeutic molecule will not only have a particular shape and form – it will have an inherent stiffness and rigidity – water molecules in solution move relatively freely and will shift to surround any molecules dissolved it them – that is why water is such an excellent solvent. But not homeopathic solutions. Oh no.

In order to circumvent this, the energetics of homeopathic remedies must be amazing – water molecules typically interact with each other via low energy, transient “hydrogen bonds” – each hydrogen bond requires 5-30 kJ/mol to break it (depending upon solvent conditions, temperature, etc), but a permanent “covalent” bond that links atoms in a drug molecule typically requires over 300 kJ/mol to break it – i.e. at least over 10 times stronger. And yet the homeopathic shell of water molecules manages to be as strong as this. Utterly fantastic stuff.

The properties of homeopathic solutions are beyond comprehension – and to think that all this was achieved by diluting something to a point where no original molecules remain whilst periodically banging it on something leathery.

Homeopathy is truly unbelievable.

It is the fantastic and unbelievable nature of homeopathic remedies that leads scientists to question their validity as an effective form of therapy. Experiments to investigate this show that homeopathy is no more effective than placebo. When a therapy has no active ingredient, no conceivable mechanism of action, and cannot be demonstrated to be more effective than a placebo, to claim otherwise is both dangerous and immoral. It certainly has no place whatsoever in a public funded health service like the NHS.


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