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Permitted E's damage our DNA

Bulgarian experts in genetics proved at least six E’s damage cells, destroy DNA and create risk of cancer, permitted dose to be decreased 10 to 100 times

We know that food additives, assigned as "E", are not a bouquet of vitamins, but do we know that some of them literally place a bomb in our organism by damaging DNA? In this way, they create a serious risk of cancer. Even worse – they can be passed down to our progeny. The most merciless killers turned out to be the colorants. Bulgarian experts in genetics have studied 20 Е's, and for 6 of them their data are definite: they introduce harmful changes to DNA, at concentration levels even lower than the ones currently permitted. In order for them to be safe, the E's have to be dosed 10 to 100 times less than the statutory accepted dosage. Magazine 8 witnessed the truth about E's live from the research lab.


Almost no packaged or canned product exists on the market, which does not include additives, also known as "E's". These additives are several groups – preservatives, stabilizers, colorants, sweeteners, raising agents, emulsifiers. Generally speaking, their purpose is to make products longer lasting, tastier, and more attractive to the eye. (We wrote in detail about E's in issue 12 of 2009 of Magazine 8). The use of several hundred E's has been approved in the European Union, and their eligible product concentration is regulated. The packaging does not mention what it is, but it is mandatory to list E's. The presumption is that they contain the permissible quantities.


And here we reach the most important question – whether those permissible quantities are harmless. No, they are not. At least the 6 E's – which include 1 preservative, 3 colorants, energy drinks additive, and a popular ingredient in analgesics – are in concentrations, which could cause cancer!   Associate Professor Doctor Georgi Miloshev – Director of the Molecular Genetics Laboratory with The Institute of Molecular Biology of the Bulgarian Academy of Science, has studied some 20 E's, and has published results for 6 of them. 

He has upgraded a method for evaluation of impact E's have on DNA. His method is 100 to 10 000 times more sensitive of the methods used until this time. Associate Professor Miloshev explains: "When the eligible quantities of various additives in foods have been determined, no one has studied their impact over DNA. DNA is the matrix of life, and is the first stage, in which damage could be caused, which could directly result in pathologies in organisms. Unfortunately, more than 30 or 40 years ago, the toxicity of food additives and preservatives has been determined with experiments on mice. It has been observed under what quantities malformations and damages to different organs of the model samples have occurred (liver, blood circulatory system, etc.), as well as when the animal gets cancer. Thus, by analogy, it has been evaluated what the safe dosage is for humans. And that was that".


Using its modernized and much more sensitive method for reporting the impact of substances to DNA, Associate Professor Miloshev has checked whether in reality the permissible concentrations of E's are safe. He studies the impact of approximately 20 additives over DNA, and manages to publish the results obtained.

What Associate Professor Miloshev and his team have studied is the impact of the studied E's over the genome (DNA). The results published so far are for one preservative - sodium nitrite Е 250; three colorants: indigo carmine Е 132 (gives violet color), erythrosine Е 127 (gives red color) and fast green Е 143 (grants green color); the additive caffeine – widely used in energy drinks, and four-amino-antipyrin (with application of the pharmaceutical industry, added to analgesics).

The scientists of Molecular Genetics Laboratory, IMB – the Bulgarian Academy of Science treat the cells with extremely high doses from these additives, go through the eligible doses, and continue with values way below the permitted doses. Thus, they check what concentration of all of these substances has caused damage to DNA. Of course, control is applied – when the cell is not treated with substance, but instead with pure water.


The result if both definitive and horrifying! "The eligible values of all studied E's have caused damage to DNA. In the various substances, damage depends on the dosage, but exists for all six E's. To be fair, I did not expect that permissible quantities will be reflected to such a horrific degree. Significant data exists that 14 other additives have the same effect", says Associate Professor Miloshev. He then explains:   

"Different types of DNA damage exist. For example ruptures in the DNA chain – in one or two strands at the same time. More powerful chemicals rupture both, which is very dangerous for the cell, because it can cause cancer! If just one of the strands is broken, it is easier to "repair" it. Another type of damage is the so-called "chemical modification" – the E's chemically modify the DNA bases (building blocks), by adding methyl groups to them. Thus they have influence over the processes in DNA: replication (doubling DNA) or the transcript (re-recording information until albumin synthesis), which as a result of the damage caused, do not occur in a correct manner. The third type of damage: the E's could cause irreversible binding of both DNA chains, so that the double helix cannot be unbound during replication, and this is the basis of cell division. All of the above-listed could cause DNA mutations, or the mutations are harmful to the organism. The substances, which cause this, are called mutagens. These are cigarette smoke, artificial fertilizers, processed vehicle exhaust gases. The E's are also mutagens. The main consequence of DNA damages is that they could cause cancer".   

The study of Associate Professor Miloshev has indicated that the hazard remains even in case of decreasing the "dosage" of the E's. In order to not have damage to DNA, and respectively – risk of cancer, the studied substances shall be in concentration 10 to 100 times less than the currently permissible. 


"Out of all E's, the ones that damage DNA the most are colorants! Colored candy children love so much are extremely dangerous, and out of the colored cakes, the ones with a photo on top are the most harmful", emphasizes the expert on genetics. The sodium nitrate (Е 250) is standard and is a very highly widespread preservative. 

It is also used in metal cans storing fish, meat, vegetables, and cooked meals. The producers prefer it, because small quantities of it work better than high quantities of salt. It is accepted to use in EU and its permissible quantities have been defined. But it's also dangerous. It affects DNA most heavily, as colorants do, and it kills the cell employing another mechanism – it damages organelles inside of it, and blocks albumin synthesis.   

Is salvation possible? With foods produced by the industry, preservatives could not be avoided. Manufacturers would hardly give up their bright colors, which make their commodities attractive.   Associate Professor Miloshev has found the precise safe values for each of the studied E's. It would be normal if producers contacted scientists and asked them: "What quantity of E's is it safe to use?" But will they listen to their opinion, if there is a possibility for them to not get the bright red color, which sells so well? And will they be willing to look for other – harmless – colorants, if this affects their pockets? "Scientists have the duty to warn the public of the risks, and the public has the right to insist on politicians and the respective institutions for such changes that would preserve public health", says Associate Professor Miloshev.   

More information available at www.epigenetics4U.blogspot.com

The studied E's: what, where, why, how much  
Compound Description Used in: Concentration (in mmol/L)
Used Safe
Sodium nitrite Е 250 (preservative) Inorganic compound. Used in the production of paint and some organic substances. In the stomach, along with other hazardous substances, it could form the cancerogene N-nitrosamine Caviar, meat, poultry and game products, which do not undergo heat processing. Also fish and fish products ready-made foods. 0.1-2.5 Under0.00001
Caffeine(additive) Plant alkaloid. Possibly pharmacologically active substance most frequently utilized during digestion in the world. Coffee, tea, Coca-Cola, energy drinks, milk chocolate, etc. 0.1-14.4 below0.001
Indigo carmine Е 132(colorant) Organic compound of specific blue color. Almost all types used today are synthetic. Jams, jellies, fruit pulps, purees, cocoa milk; cocoa and chocolate products, gums, bakeries; fresh fish; this is the colorant, which shall be used in gastrointestinal endoscopy. 0.1-4.3 below0.001
Erythrosine Е 127(colorant) Synthetic paint for coloring food products. It is also included in the composition of certain inks of the printing industry. Dried and sweetened fruits, ice creams, sorbets, jams, jellies, fruit semi-processed foods, hard and soft candy, chewing gums, pastry articles, etc. 0.05-0.5 below0.001
Fast Green Е 143(colorant) Synthetic dye for coloring food products . Canned and bottled fruit juices, pickles, sauces, semi-prepared pasta (spaghetti, etc.), liqueurs, beer, wine, desserts, with high concentration of eggs. 0.1-0.75 below0.001
4-amino-antipyrin (4-AAP)(additive to analgesics) Widespread application in pharmaceutical industry. It is considered that in the cell, 4-AAP is modified in a manner, which could cause damage to DNA. Metabolite of various medicinal substances. (Included in the composition of aminopyrine, metamizole, non-steroid anti-inflammatory medicines). It has analgesic, antipyretic and anti-inflammatory effect. 0.5-8.4 below0.01

The damaged double helix of life now looks like the tailed space body, and forebodes illness and death

Twenty minutes. The lapse of short period of time, which feels like a century. This is the span of time for organizing an important experiment. The influence of preservatives, colorants and improvement agents, with widespread use in the food industry, over the DNA molecule. The experiment is conducted by the scientists of the Molecular Genetics Laboratory with the Institute of Molecular Biology – the Bulgarian Academy of Science. All of them are professionals in this field, but despite this, each experiment is a thrill for them.

The researchers are still waiting, and they are getting impatient and display curiosity openly toward another mystery of Nature. The test results are reviewed with a microscope. Associate Professor Doctor Georgi Miloshev, Director of the Laboratory, keeps staring at the eyepiece, and all other team members have gathered around him. In the stony silence, all that can be heard is their breathing. The director looks up from the microscope and barely smiles. The team erupts in cheers. "Yeah!" Was it really like this, we ask Associate Professor Miloshev. He bursts into laughter: "Well, not quite, but there was excitement". The scientist has just told us of, and has shown us the stages of the study on the effect of the food additives, known as E's, over DNA. Following his recount, we mark the fifth stage of the study as the most dramatic one. We name the next one "the moment of truth". The team of Magazine 8 observes all stages, which took us a couple of days. But this is just the "summary version" of the whole story; as we observed the manner that the method is applied just for a single type of E's (colorants), and only in single concentration.   


The real story follows the same stages, but they are repeated hundreds of times – for several types of E's, studied in different concentrations, and each "sample" is repeated multiple times. The project takes the team three years to complete.

The conclusions of the study have been already reported to you in our first text (page 38), and now we are going to show you exactly how things transpire in the laboratory on discovery of E's as killers. The method that scientists apply in this case has been used to determine the quantity and the type of damaging of DNA, caused by the additives and the foods, marked with Е. But using this method, all types of "suspicious" substances could be tested, which have access to live cells. Therefore, it is applicable in studying air, paints, cosmetic products, even for finding radioactive contamination. It is called "Comet test", because unlike integral DNA, which remains unaffected in a cell nucleus, the damaged DNA actually goes out, and can be observed as a comet with a tail under a microscope. In this case the test is applied over cell culture of yeast, because they are accessible and very sensitive to chemicals. The test, however, has also been applied on human cells, and then the results have been compared. Its validity matches, because regardless of whether it is human a human cell or not – it is structured and functions in the same manner, and is affected by chemical agents. The method has been known for a long time, and is applied widely. However, Associate Professor Miloshev has upgraded its design in such a manner, that it has made it much more sensitive.   

For the layman, Molecular Genetics Laboratory is a jungle of machine with wires, pipes, jars, all different kinds of vessels, boxes and pipettes. The members of the team in white aprons Milena Georgieva, Desislava Staneva, Borislava Boteva, Toni Efremov and Juliya Milcheva navigate this jungle with such certainty that they seem to be at home in it. With the slightest movement, Milena manages to dose just a single drop, and the pipette is obeying her, like it's a natural extension of her hand. No nerves or pressure is felt. "This is what happens you're your job is also your hobby", they all say.


And so, we start with the research protocol. We will bear witness on both E's impact on DNA – the food product colorants – blue and red. During the first stage of the experiment, cells are placed in a food-rich environment (it contains in general water, sugar, and protein extract), and for the duration of 2-3 hours they reside in an equipment which provides them with the optimum conditions for growth – 30 degrees and aeration. 

The purpose of this stage is under optimum cell development conditions to have them multiply up to a specifically designated number, which is considered to be 100%. Compared to it, the percentage of cells with DNA damaged by the E's shall be determined. Later, during the final stage of the study, following the statistical processing of data by specialized software, scientists shall verify this percentage.   

During the second stage, a chemical is added to the cells, while they are still in the nourishing environment, the effect of which is being studied – in this case Е-colorant. It is in powder form, and all producers buy it in this form. It is soluble in water. This solution of Е has a specific concentration. The experiment is conducted with different concentrations – concentration statutory accepted as safe, higher than the statutory accepted concentrations, and several ones, which are much lower. 

The study is repeated with a gradually decreasing concentration of the solution of the E, in order to inspect concentrations up to 10 or even 100 times lower than the statutory accepted one. Control sample is also taken – at this stage no solution of E is added to the cells, instead fresh water is added, the same quantity as in the solution of E in the other flasks. In order to be certain of the results obtained, scientists conduct the study for each concentration multiple times.


The goal, at this stage of the study is to determine the limit, below which the substance stops having a harmful effect. Under this so-called "threshold value", there is no DNA damage, but above it – there is.  Scientists have found that for certain substances the threshold value is 10 times lower than the currently accepted as statutory value, and for others – 100 times. I.e. even multiple times lower than the current quantity of Е is still damaging to DNA. 

The third stage of the study is when the so-called "admixture" of cells and E's is returned for cultivation to the equipment, which supports the optimum conditions for cell life, and constantly shakes them. This continues for hours on end. The purpose of this is to verify whether, or how, the substance added (in this case the respective Е) will affect the viability, growth, and reproduction of the cells. The scientists have established that the colorants have an excruciating effect over the viability of cells – in some concentrations they have such a strong cytotoxic effect, that between 50 and 70% of the cells die. The strongest negative effect is the effect of indigo carmine (Е 132), erythrosine (Е 127) and especially of fast green (Е 143). (Indeed, the cytotoxic effect of erythrosine is studied for the first time within the framework of this study of E's).   

Fourth stage: the cell culture, to which an E has been added, is mixed with a rapidly polymerizing gel. A specific quantity of this admixture, measured with a pipette, is dropped down over the slide, in order to "spread". Another slide is put on top in order to seal it. Between the two slides, gel is distributed evenly into a regular film. In it, DNA moves under the influence of electric field in the next stage of the study – DNA electrophoresis.   


The fifth stage is the stage we named "dramatic". During this stage, the slide with the gel (the covering slide has been again removed) is placed in a special apparatus for DNA electrophoresis for 20 minutes. During this process, and under the impact of the electric field, if there are ruptures in DNA, it will move to the plus pole. That is why it can be observed stretched like a comet tail, whereas intact DNA remains within the nucleus. Will this effect occur? For the researchers, which as of this time still cannot see the result, it is clear that these are the critical 20 minutes, during which DNA will behave in a very precisely specified manner, depending on whether it has or has not been damaged. Therefore, for scientists this period of time is the period of waiting impatiently.

  During the sixth stage, the slide is taken out of the DNA electrophoresis apparatus and is submersed in a special solution in order for the gel to be fixed. In this manner, it could be stored for years, and researchers could use it to repeat the analysis, and if anyone was interested – they could with their own eyes the degree of damage or survival of DNA.


During the seventh stage, the slide with the fixed gel is placed in a special microscope in order to observe whether DNA is in the nucleus, or it has extended like a comet. This is the moment of truth, when the researchers can see what happened with their own eyes for the first time.   

Data processing is the final stage of the study. The methods employed as of this time have determined the percentage of the damaged DNA. However, the team of Associate Professor Miloshev has developed special software, which has an additional function – just by looking at the comet it can determine the nature of the damage – rupturing of one or both strands. Most DNA damage is hazardous for the cells and the organism in general, especially when damage occurs for both strands. This could lead to the cell dying, or could cause cancer. "Our work has unambiguously proven that the E's we are studying are genotoxins. They damage DNA by rupturing it, or by modifying various chemical groups inside it. The DNA modifications can be passed down to the next generation unseen, and result in accumulating damage to it as well. In our study, we have observed it directly – Milena Georgieva has observed under a microscope which DNA is healthy and has remained whole in the cell nucleus, and which one has been damaged, and has left the nucleus.

The additives are harmful to both the current and the new generations of cells, and various E's have a different degree and manner of influence", says Associate Professor Miloshev. Except for genotoxicity, E's have been studied to confirm that they are cytotoxic and that they suppress the growth of cell culture. Both have been confirmed. 

But still, which was the most exciting moment of the whole study? "Impatience is highest while we are planning and conducting new experiments, because we do not know the result. But in this case with the E's it was not exactly like that, because I already knew they were harmful. The surprise was not that, but the scale of damage caused by even minimum quantities of Е, which we tested. It is very exciting when we come across something new and interesting, or when we have been able to understand a process. And of course pleasure is always extreme when we discover a hitherto unknown phenomenon, which allows a new direction for reflection, and a field for more experimentation. The reason for this is because a scientist is a person with great curiosity towards nature. If this is not the case, then this person is not a scientist", says Associate Professor Miloshev.