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In this lecture, I will discuss some of the aspects of free radical theory as it involves aging and cancer. The initial aspects of the free radical theory were described by Harmon in 1956 – a physicist quite familiar with the effects of radiation, who postulated that DNA damage as well as damage to other subcellular structures might be related to free radical generation and be related to aging. Free radicals range from a host of compounds including a number of compounds in the reactive oxygen species (ROS). Compounds such as the hydroxyl group can interact with free electrons and cause significant changes in the subcellular building blocks. Subsequently it has been shown that 95% of all oxygen utilized in the body are involved with a subcellular structure known as mitochondria. Much of this lecture has to do with mitochondria, since these structures turn out to be critically important in our energy production at the cellular level, as well as the generation of these free radicals. There are a number of protective enzymes in the body that are designed to absorb the free radicals including catalase and superoxide dismutase. Additionally, a number of antioxidants are present and can ward off the negative effects of these free radicals. Part of the postulate of this lecture is that the production of free radicals by mitochondria can lead to subcellular damage; subcellular damage of this type can affect DNA in the nucleus – and lead to cancer. Subcellular damage in the cytoplasm can disrupt the cellular function and particularly attack the mitochondria themselves. The development of weakened mitochondria may well play a major role in aging itself.
One must stop and look at the story in regards to mitochondria in eukaryotes. Uniformly, these structures are present in the higher life forms and account for a far more active energy process than is typically available through glycolytic pathways well known in the lower life form prokaryotes. These lower life forms do include bacteria, which represented the dominant life force on earth until approximately 2,000,000,000 years ago. Geologic evolution need be juxtaposed on the development of mitochondria as part of the cell structure itself. It is quite remarkable that in the protozoic era, there was an intermediate oxygen level possibly accounting for 5-18% of the current oxygen level. Prior to that time, earth existed primarily in a reducing atmosphere including nitrogen, sulfa dioxide, CO2, H2O, hydrogen and ammonia. The remarkable turn of events that started with increasing oxygen are well documented in sulfur studies showing various oxygen levels in pyrite (FES2). As the pyrite shows exposure to oxygen, it changes form and led to layering, showing that parts of the ocean had high and low oxygen states beginning to coexist. Most of the oxygen production was related to the remarkable success of cyano-algae, which were able to use existing reducing substances and subsequently release oxygen. The success of the cyano-algae ultimately led to the Cambrian explosion, which occurred approximately 500,000,000 years ago. This time frame in geologic and subsequent life history was associated with a dramatic rise in oxygen and a similar dramatic rise in the number of available life forms that were present. Far more complicated life forms could be present with this higher oxygen state, in the setting of increased energy availability through oxidative phosphorylation – the basic process giving energy within mitochondria. This is also the time frame in which multiple species reportedly evolved and increased predator action as the basic structure of the eye developed. I have read remarkable suggestion that oxygen levels may have exceeded 21% and even moved to a level much higher, possibly 40-50%, peaking at the time of the great dinosaurs. In this time frame, very large animals as well as insects existed including, as an example, the dragonfly – with a 1 meter wing span. It has been postulated that the higher level of oxygen allowed such giant creatures to exist. Additionally, one tantalizing suggestion is that the asteroid reportedly destroying the dinosaurs and many of these life forms that fell 65,000,000 years ago destroyed the life forms through a massive oxygen firestorm, rather than just simple dust occluding the ability of sunlight to penetrate. In any event, we have in this time frame moved to a stable situation in which oxygen is our friend and subcellular enemy at the same time.
It is important to reflect on some of the issues of endosymbiosis as to the origin of mitochondria in cells and life forms as we know them today. Mitochondria are completely unique as to subcellular structures, in that they have their own unique DNA. All mitochondria trace their origin to a single ancestral protomitochondrial genome. It is felt that ancestral prokaryotes – bacterial derivatives – included primarily anaerobic energy, the waste products of which could be utilized by proteobacteria. We have in the world today good examples of endosymbiosis such as rickettsia – a disorder associated with typhus fever. In that disorder, the organism is simulated into the cell, infects and survives utilizing the metabolism of that cell itself. In the 1960’s and 1970’s, the possibility that early life represented a symbiosis of a rickettsial-like organism and a proteobacteria was postulated. As evolution is reported to continue, some of the genome from these early mitochondria-like forms is reported to have migrated to the main nucleus of the cell. Of note, none of the main nucleus genes have ever been felt to be migrating into the mitochondria themselves. In spite of some of the transmigration of mitochondria genome into the cell nucleus, the mitochondria itself is quite unique. The unique nature of mitochondria includes that it is the only cellular organelle with its own genome. This genome is quite small, representing approximately 16.5 kilobytes of information in nucleotides as opposed to the 3,000,000,000 base pairs seen in the main nucleus. Unlike the format in the nucleus, a mitochondria DNA is circular in character and contains information for 22 transfer RNA, 2 ribosomal RNA, and 13 polypeptides. The entire mitochondria has no additional DNA present, as opposed to the main nucleus. In the main nucleus, the exons represent the part of DNA that is known to make proteins. This accounts for only 5% of the DNA and the other 95% is DNA that includes satellite DNA (now used in cases such as the O. J. Simpson trial), as well as jumping genes such as transposons, as well as retroviruses. All of the genes for peptides in mitochondria are directly related to the stages that are necessary for electron transport system associated with oxidated phosphorylation – the main powerhouse to the cell. Every nucleated cell in the body has its own mitochondria to act as an energy source as the cell grows.
Interestingly, there are 2-10 copies of mitochondrial DNA per mitochondria itself. Additionally, every cell has anywhere from 10-2,000 mitochondria present. The mitochondrial profile for any given cell type is unique to that cell type. Some are in the neighborhood of 400-500 cells described in humans and felt to be up to 1,500 types of cells, many of which have not yet been described. All of the mitochondria in an adult comes from the cytoplasm of the egg – all mitochondria come basically from the mother. The mitochondria that are associated with the sperm are destroyed when the union of egg and sperm occur. This destruction process is directly interrelated to activation of the ubiquitan system. Ubiquitan system is necessary in cellular function as a general housecleaning mechanism for ridding the cell of proteins that are no longer needed. In the main nucleus of the cell, there are approximately 150 nuclear peptides, which are coded and act as “chaperones”. These chaperones facilitate transmigration of various products needed for mitochondrial function and allow passage across the mitochondrial membrane into the lumen of the mitochondria for subsequent usage. Most importantly, mitochondria have no histones and no introns. Histones are remarkably important in DNA, since they are responsible for turning on and off DNA – not all the DNA that can make a protein is turned on at any time. The inactivation of DNA through the mechanisms of histones is modified by various components of the diet, endocrinologic system, and other subcellular mechanisms. The introns that are associated with the cellular nucleus are the so-called satellite DNA that is associated with forensic DNA testing. Lastly, DNA in mitochondria are particularly susceptible to injury and have very limited opportunity for repair, since none of the proteins made in mitochondria are designed for DNA repair. It is a very important concept that DNA in mitochondria are therefore very sensitive to oxidative forces. The oxidative forces are also particularly prevalent inside the mitochondria – since most oxidative forces are associated with oxygen metabolism and 95% of all oxygen metabolism occurs in the mitochondria. Satellite DNA or introns act as a protective mechanism in the nucleus itself. The nucleus uses these structures to accept oxidative stresses and divert the oxidative stresses away from the exon – protein forming DNA – and into the less important satellite DNA component. It is also very interesting that mitochondria have a unique codon language. The codon language refers to combinations of three bases and their subsequent interpretation as to which amino acids they would represent. Mitochondria need their own transcription RNA, because they don’t speak the same language as the nucleus. In an adult human, there would most likely be upwards of 10,000,000,000,000 cells, each of them having need for active mitochondria per energy production. Just like the entire electrical grid in the United States has no major way of storing the electricity, the cell is dependent on an ongoing source of electron transport to generate energy for cellular activities. This makes it quite critical that the mitochondria be active and functional, and as part of the unifying process that aging is associated with mitochondrial dysfunction from oxidative forces within the cell and exogenous to the cell. These oxidative forces can result in a unique process known as hetereoplasmy amongst the mitochondria. That is to say that a given cell, as it ages, has the mitochondria develop changes in the DNA of these mitochondria unique for that cell line. In that setting, the damaged or changed mitochondria will have different function and this will be characteristic of that cell line alone. This also would potentially account for differential aging, where one may see a patient who has only one system that shows accelerated aging and the rest of the patient looks just fine. Oxidative forces account for changes seen in numerous aging-related diseases, whether they are cataracts, Alzheimer’s disease, atherosclerosis or cancer. The differential aging components in the cell lines, therefore, will account for the combination of aging process as well as cancer itself. I make the latter statement, since it has become apparent in the last two decades that cancer represents a change in the actual genome for a particular cell line. Whether it is a breast cancer, colon cancer or pancreatic cancer, etc., all of these cell lines, when plagued with cancer, have DNA changes that are unique to that cell line. Again, that would represent a cumulative effect of oxidative forces with the subsequent end-stage being a cancer “phenotype”. Loss of DNA fidelity, therefore, represents a very important step in the actual development of cancer, and one can postulate that the oxidative forces that are associated with this loss of fidelity are quite critical in production of this type problem.
Let us return for a moment to the presence of mitochondria as exceptionally sophisticated organelles skilled at the use of oxygen to generate energy for the cell’s benefit. If mitochondria became incorporated into eukaryotes approximately 2+ billion years ago, it is difficult to understand how any form of evolution would have allowed this unique presentation in an environment that was low in oxygen at that time frame. It has been suggested that if an external force, whether creation or delivery of mitochondria to earth occurred, the process may well be the best explanation for how such a sophisticated organelle can exist and begin endosymbiosis in a time frame in which oxygen was not a critically important component to the atmosphere. I suspect that since most meteors come from Mars, one of the best reasons for the current Mars Rover Expedition would be to look for evidence of not just water, but possible life forms that would herald the process of transplanetary DNA transfer that might be associated with migration of mitochondria to the Earth. Otherwise, one has a difficult time postulating such a sophisticated set of enzymes evolving with no stress factors or opportunity to deal with oxygen in the early Earth stages. Certainly this concept is well beyond the scope of this particular lecture, but raises some interest level in even the most casual observer. In April of 2003, we are at the 50th anniversary of the description for the function of DNA as described by Watson and Crick. This terrific insight has resulted in an explosion of information in the past 50 years as to how life works and the actual building blocks necessary for such function. Though they worked out the structure of DNA, they were somewhat remiss in not discussing the additional information that the actual structure of DNA allows for a “backup” copy. In the time frame of the original description, DNA was mostly known in the crystalline state, in which it is quite stable. We subsequently know much more about DNA and find that it is very sensitive to minor oxidative forces. The repair and backup system for DNA did not occur to Watson or Crick, and yet DNA repair turns out to be fundamental to life itself. Certainly most of the oncogenes that are now described turn out to be repair genes that are defective in some fashion, thus giving an increased risk to the organism cell line to develop a cancer. Loss of DNA repair, therefore, is associated with loss of DNA fidelity and is the basic process often seen in aging and cancer itself. There are at least 130 human genes that are associated with DNA repair. Many of the most famous oncogenes are, in fact, DNA repair genes. If one is born with such a DNA repair gene problem, an individual will have a marked increased risk of developing a cancer in affected cell line. A nice example would be associated with BRAC genes in breast cancer, but certainly it can be found in almost any type of cancer that a genetic tendency exists, and is almost always based on problems related to DNA repair. It turns out that an intrinsic metabolism, since water is a critical component of this system and water contains oxygen, there is a staggering barrage of oxidative events in every cell in the body. It has been estimated that there are at least 30,000 such events per day per cell and given the presence of 10,000,000,000,000 cells, it turns out that DNA repair is a really critical process. These oxidative events may come in the form of intrinsic metabolism, but also are associated with exogenous cancer-causing agents, whether dietary or from agents such as smoking and pollution. The subtleties of these changes lead to effects many years later, as is now being seen with Agent Orange and the risks that we have associated with dioxins, etc., in our own environment here in Escambia County, Florida. It is of some note that these risks cannot easily be measured, but there are mechanisms now available to look at oxidative forces that have affected the DNA. Most specifically of note is that guanine, one of the nucleotides critical for DNA, is particularly susceptible to oxidative events. The particular ring structure of guanine tends to donate an electron much easier than the other nucleotides, and in the presence of a hydroxygroup can make a compound known as 8-oxoguanine. 8-oxoguanine can be measured and found to reflect the underlying oxidative stress on DNA, and one would report that this would be an excellent screening mechanism for those who are subject to unusual oxidative stresses, i.e. living near a super-fun site or smoking, etc. The overall internal oxidative state of your DNA, whether of mitochondrial origin or nuclear origin, turns out to be remarkably important and I would suspect that commercial tests at some point will be very common to assess a person’s oxidative risk – which will be directly proportional to their risk of cancer and aging events. This would also be particularly useful in sleeping, in regards to environmental toxins, etc. Other type screening mechanisms can also be postulated as a role, since antioxidants such as glutathione can be measured and estimated as to their reduced or oxidized state, and therefore extrapolated as to the overall cellular oxidative mix that is present. All of these represent a likely burgeoning process in the future, once such technology can be applied clinically and its utility confirmed to the general populous. It is also interesting to note that guanine in some ways can 0 be protective of the genome itself. It is very surprising to now find that there has been some form of selective pressure to put high numbers of serial guanines at the interface between an exon and intron. This interface is quite important, since it has been found that DNA will conduct electricity. In fact, the DNA ability to conduct electricity as well as its very specific nature is probably the reason why there is quite a bit of excitement that at some point DNA may replace silicone in the future computer chips, to allow Moore’s law to continue – the law that states that we will continue to have an increasingly shrinking chip option in computers of the future. After all, only at the molecular level can the final limits be reached for control of that type process. In any event, the guanines are serially stacked right at the interface because guanines, when in a row, will act as cathode protective sites. This cathode protection is exactly the process that is associated with the discovery in 1824 that small amounts of zinc can protect an iron hull from rusting.
Returning to the mitochondria then, we find that the lack of protection for mitochondrial DNA make mitochondria particularly susceptible to oxidative forces. It has been found that mitochondrial DNA show anywhere from 2-15 times as much evidence of mutation as compared to the DNA that is in the cell nucleus itself. As mentioned, this leads to damage within the mitochondria themselves, but since each cell has different exposures to oxidative forces, it leads to unique patterns of mitochondrial change. It has been shown that cancer cell lines have particularly prominent mitochondrial pattern changes, and again the mitochondria in these cancer cell lines often have DNA with far more mutations than the actual nuclear DNA shows. It has 1 become apparent that certain underlying diseases have accelerated aging; these are in the family of progeria. Interestingly, last summer the genetic cause of progeria of the typical type was identified and found to be a component of mitochondrial membranes. The notion is that these abnormal lamellar proteins are associated with increased leakage of reactive oxygen species – which leads to a far accelerated rate of aging. Additionally, there has been a series of studies on centerians. These remarkable individuals do show changes in mitochondria that suggests that their membranes are more stable, and therefore they leak less in the way of reactive oxygen species and this, at least in part, accounts for their remarkable longevity. Utilization of the mitochondria – and over-utilization – therefore result in reactive oxygen species. It is of note that in the New England Journal of Medicine in April of last year, an important study came out entitled “Overweight, Obesity, and Mortality from Cancer in a Cohort of U.S. Adults”. Basically, most forms of cancer could be found to be increased in patients showing marked obesity. One may postulate that the mechanism of this observation is that those who are obese have overutilized their mitochondria that is increasing the amount of reactive oxygen species. Though this possibility was not discussed in the paper, it is apparent that other papers have shown that test organisms, whether mice, drosophilae or C. elegans, the worm, have protective benefit if they are fed specific diets. Diets that are low in carbohydrates turn out to be particularly effective in decreasing risk, and it is felt that reactive oxygen species may be more prone if a main energy source is carbohydrate. Additionally, cutting the caloric intake of almost any organism to approximately a 60% level from baseline has been resultant in elongation of 2 lifespan. All of these test species now seem to show that decreased energy actually leads to longer life span, and it is entirely possible it is interrelated to utilization of the mitochondria, again that you have overrun your battery and resulted in too much leakage of reactive oxygen species. Therefore, the fed state of the organism helps determine its risk factors, whether it is for aging or cancer, and this certainly is backed up by many type studies. It also leads us to speculate on what possible beneficial effects one may consider from a dietary standpoint. Certainly I have already alluded to the total fed state being important, where obesity and overeating as well as high-carbohydrate diet may be associated with increased risk of reactive oxygen species. Additionally, fasting significantly with an overall lower caloric intake would be of benefit. From the standpoint of antioxidants, it is apparent that certain foods have polyphenols, most notably wine, and one of those is known as resveratrol, which is present in red wine. These agents apparently act as antioxidants and interestingly, the red wine compound is postulated to have such benefit that a no-alcohol wine extract is now being marketed and tested as an antioxidant compound. Interestingly, the membrane-stabilizing component of flavones and squalenes are most notable in olive oil. Olive oil is felt to be the reason why the Greek population has such a low incidence of cancer in breasts, amongst all the Caucasian background – followed only by the Italians. Apparently, Greeks eat two ounces a day of olive oil, where Italians have an ounce a day, and it would appear that if one is interested in the breast that olive oil is to be encouraged. Dark chocolate is associated with a compound known as epicatechin and this has been found to be an antioxidant, and the good news is that it may be good for you. Other agents one might postulate include NAD, which is a cofactor in reduction phase and has been 3 shown in animal models to be protective against oxidative stresses. One also might consider taking additives such as glutathione, vitamin C, vitamin E, and carnitine. Oxidants that are likely to be of a negative value include Co-Enzyme Q, which increases the oxidative metabolism in certain test animals and has been shown to shorten the life span, similar to overeating. Additionally, alcohol represents a significant oxidant and may account for the actual mechanism of oxygen damage associated with this compound. My suspicion is that the recent flurry of interest in the DAF-2 gene that is associated with an Insulin-like factor and has a relationship to the lifespan of the organism is more involved with stimulus of the carbohydrate pathways and the exposure to reactive oxygen species from this pathway. I have seen reference that future athletes may wish to have increased DAF-2 or other carbohydrate-based receptor genes placed so that they can have increased strength. One would probably better postulate a vector that will increase the amount of superoxide dismutase or catalase, both of which are enzymes critical in the pathway for oxidative stress.
Another risk factor that is of interest is that Eskimos have a low incidence of agingrelated diseases such as Parkinson’s and Alzheimer’s. It is felt that the unusual component of their mitochondria is that they tend to generate heat – because it is cold up there. Mitochondria can be blocked in their activity for standard metabolism with so-called uncoupling agents. I have previously summarized the activity of uncoupling agents in regards to obesity in my obesity lecture available on our web site (www.endo-world.com). Pharmaceutical houses are interested in uncoupling agents, since they can block the actual activity of mitochondria and thus releases 4 from the burden of overeating. Some of these uncoupling agents are associated with heat production instead of high-energy production. Certainly heat production might be beneficial, since less of the energy is going into reactive oxygen species and causing less stress to the organism, i.e. the Eskimo.
Some somewhat obtuse observations from reading include some additional observations in regards to the electrophilic nature of DNA. It turns out that the DNA repair enzymes, using a combination of sulfur-iron metallo complex can send an electrical signal into the DNA. These repair enzymes then send back whether the charge is taken up in the DNA, suggesting that an oxidative event has occurred upstream and that repair will be necessary. Apparently, the fact that DNA can conduct electricity 60-100 bases would allow the repair enzyme to stay coupled to the DNA stand itself, or release itself and find a more likely area for repair. Workers at Cal Tech right now are working on very exciting prospects for evaluating these electric pulses sent out by enzymes. Speaking of electric pulses, it is very interesting that a recent article suggested that nanopulses of high energy may be beneficial in treatment of tumors. Apparently, the method involves exposing cells to an extremely powerful electric field, but for an extremely brief moment. Tumor cells apparently are particularly sensitive to this pulse, and this activates the mitochondrial caspase system. I have not spent much time discussing the very critical role that mitochondria have in apoptosis, but they are central to the entire cascade that is related to controlled cell death. Activation of the caspase system can be instituted by nanosecond pulses of broadband radio signals, and represent a future potential treatment for cancer-type cells. I am 5 suspicious that the differential sensitivity of cancer cells is potentially interrelated to the underlying weakness seen in cancer cells in regards to their mitochondria. The burden of abnormal mitochondria in a given tumor line generates a completely different profile for energy activation in that tumor line. Additionally, the new scan known as a positron emission tomography (PET) scan works on the same process. In PET scanning, it is found that the mitochondria have an increase in the less active glycolytic cycle, possibly because their mitochondria are defective. In any event, the use of a fluorinated glucose derivative can light up a PET scan because of the increased sensitivity in tumor cells to the glycolytic pathway. The entire electrical field around the mitochondria must be influx and quite sensitive, and with the nanosecond radio wave system, one can anticipate a treatment modality similar to the PET scan based on underlying mechanisms involving the mitochondrial electrical milieu. It is interesting that one can increase the effect of radiation by giving specific modified bases to the organism before radiation. One such base is a bromo-uracil compound; the bromo-uracil compound almost certainly plays a role in the radiation exposure through the increased sensitivity to lowenergy forces to activate the uracil and make it more toxic to the tumor cell. The electrophilic nature of the ring structure in uracil represents the same process I have described in the example with cathode protection component.
The last interesting example is that life span has been felt to be affected with a limited number of cell duplications possible in a given stem cell line. This is felt to be on the basis of telomere length. The telomere length reflects the number of divisions a cell can go through before it no longer has the free nucleotides on the tip that allow division to occur. Quite a stir 6 was spread several years ago when the Geron Company announced that it had developed a reported immortal cell line, because they had reintroduced telomerase into the system and thus allowed the cell to have more divisions than otherwise likely. Entire relation, however, between aging and cancer is such that I am not sure that such a step could ever be clinically active in humans. The most telling aspect is that the animal models have shown that the rate of telomere change per year, rather than the overall telomere length is the best predictor of maximum life span. The so-called Hayflick limit may play a significant role in that the number of possible divisions is limited not just by telomere length, but also by mitochondrial activities. All of these reflections suggest that there is a new field opening to understand the underlying aspects of aging and cancer. It will not be surprising to see if genetic engineering can address ways of stopping leaking mitochondria, giving aging a positive note. It certainly will allow better insight as to the dietary suggestions that can be made, as well as very important insight as to the risk factors associated with various environmental studies that are currently undergoing very important scrutiny here in this country.
Roger M. Orth, M.D.