Archive for the ‘Molecular Genetics’ Category

Ancestors Of African Pygmies And Neighboring Farmers Separated Around 60,000 Years Ago

Jan 17th, 12 / 0 Comments

Ancestors Of African Pygmies And Neighboring Farmers Separated Around 60,000 Years Ago

Etienne Patin, et al. (2009) PLoS

ScienceDaily (Apr. 11, 2009) — All African Pygmies, inhabiting a large territory extending west-to-east along Central Africa, descend from a unique population who lived around 20,000 years ago, according to an international study led by researchers at the Institut Pasteur in Paris. The research concludes that the ancestors of present-day African Pygmies and farmers separated ~60,000 years ago.

Pygmies are characterized by a forest-dwelling hunter-gathering lifestyle and distinctive cultural practices and physical traits (e.g., low stature). Two groups of Pygmy populations live in the African rainforests: the “Western Pygmies” and the “Eastern Pygmies”. The common origins of the two groups of Pygmies, separated by thousands of kilometers, have been long debated, and their relationships with neighboring farmers remained obscure.

The researchers, led by Lluis Quintana-Murci, studied the genetic profile of twelve populations of Pygmies and neighboring farmers dispersed over the African continent, using sequence data from non-coding regions of their genomes. Using simulation-based procedures, they determined that the ancestors of Pygmy hunter-gatherers and farming populations started to diverge ~60,000 years ago, coinciding with a period of important human migration both within and outside Africa. Much later, ~20,000 years ago, Western and Eastern Pygmies separated, concurrently with a period of climate change leading to large retreats of the equatorial rainforest into refugia.

The common origin of all Pygmies unmasked in this study led Etienne Patin, one of the leading authors, to conclude that “they have probably inherited their distinctive shared physical traits, such as low height, from a common ancestor, rather than by convergent adaptation to the rainforest”. However, complete genome-wide profiles of these populations are now needed, both to characterize more precisely their demographic history and to identify genes involved in the adaptation of these populations with different lifestyles to their specific ecological habitats.

Hunter Gatherers, Molecular Genetics, Paleoanthropology

Ancestors of African Pygmies Separated 60,000 Years Ago

Jul 10th, 11 / 0 Comments

 

ScienceDaily (Apr. 11, 2009) — All African Pygmies, inhabiting a large territory extending west-to-east along Central Africa, descend from a unique population who lived around 20,000 years ago, according to an international study led by researchers at the Institut Pasteur in Paris. The research concludes that the ancestors of present-day African Pygmies and farmers separated ~60,000 years ago.

Pygmies are characterized by a forest-dwelling hunter-gathering lifestyle and distinctive cultural practices and physical traits (e.g., low stature). Two groups of Pygmy populations live in the African rainforests: the "Western Pygmies" and the "Eastern Pygmies". The common origins of the two groups of Pygmies, separated by thousands of kilometers, have been long debated, and their relationships with neighboring farmers remained obscure.

The researchers, led by Lluis Quintana-Murci, studied the genetic profile of twelve populations of Pygmies and neighboring farmers dispersed over the African continent, using sequence data from non-coding regions of their genomes. Using simulation-based procedures, they determined that the ancestors of Pygmy hunter-gatherers and farming populations started to diverge ~60,000 years ago, coinciding with a period of important human migration both within and outside Africa. Much later, ~20,000 years ago, Western and Eastern Pygmies separated, concurrently with a period of climate change leading to large retreats of the equatorial rainforest into refugia.

The common origin of all Pygmies unmasked in this study led Etienne Patin, one of the leading authors, to conclude that "they have probably inherited their distinctive shared physical traits, such as low height, from a common ancestor, rather than by convergent adaptation to the rainforest". However, complete genome-wide profiles of these populations are now needed, both to characterize more precisely their demographic history and to identify genes involved in the adaptation of these populations with different lifestyles to their specific ecological habitats.

 

Journal Reference:

  1. Patin et al. Inferring the Demographic History of African Farmers and Pygmy Hunter-Gatherers Using a Multilocus Resequencing Data Set. PLoS Genetics, 2009; 5 (4): e1000448 DOI:10.1371/journal.pgen.1000448
 
 
Hunter Gatherers, Molecular Genetics, Paleoanthropology

The Empire Strikes Back

Jul 9th, 11 / 0 Comments

 

                                    “THE EMPIRE STRIKES BACK”

                  How Agribusiness Treats Scientists Who Question GE Safety

                                             by Ken Fischman, Ph.D.

 

Doctor Pusztai’s Potatoes

     My Phone was ringing off the hook.  When I breathlessly reached it, I found my neighbor, Dr. Charles Benbrook on the other end.  “I have a house guest who I think you might want to meet” he said.  “It’s Dr. Arpad Pusztai. We are having a get-together tonight at my house.  Do you want to come?”

Did I want to come?  Is the Pope Catholic? Do cows give milk?  I had been reading about Pusztai for months in preparing for a lecture I was about to give on Genetic Engineering.  The name Arpad Pusztai (pronounced poos-tee) is not exactly a household word, but in some rarified circles he has rock star status.  He lives in Scotland.  What on Earth was he doing in the little town of Sandpoint, Idaho? I had better begin at the beginning.

Genetic Engineering(GE) is the science of taking genes from one organism and inserting them in the cells of another, thus making novel combinations of genes that never would have appeared in the normal course of Evolution.  e.g. When a gene for producing the pesticide Bt is inserted into corn, every cell in the corn plant becomes a miniature factory.

Right from the beginning, there has been controversy about the nature of these new combinations, dubbed Genetically Modified Organisms (GMOs) and their possible effects. Proponents of GE, which include Agribusiness, many Molecular Biologists (scientists who do GE), and government agencies, argue that it will bring great benefits and is safe because GMOs are essentially the same as naturally-occurring organisms.  Its opponents disagree, both as to the so-called “normal” nature of GMOs and as to their possible effects.

The question arises, how can we determine if GMOs, are safe to use as food and to let into our environment? One obvious way is to do scientifically controlled experiments on their safety.  But because of the official government attitude that GMOs are a priori “substantially the same” as natural organisms, relatively little research into that question has been done.

In 1998, Arpad Pusztai, who worked in the Rowett Institute in Edinborough, Scotland, received the first grant in the United Kingdom to examine the effects of GMO food on animals.  Dr. Pusztai, who fled his native Hungary during the anticommunist uprisings of the 1950s, is a biochemist, who specializes in nutritional studies.  He has written almost 300 scientific papers and has an international reputation.  He was thrilled to get the grant.  He did not know that it was going to destroy his scientific career.

Dr Pusztai studied rats fed GMO potatoes, in which a gene from the Snowdrop plant was inserted.  That gene produces a Lectin.  That is a chemical that helps protect plants from insect pests.  He thought that it was going to be a straightforward study that would support the conventional scientific wisdom that GMO plants were just like ordinary plants.  He found instead that the presence of the gene resulted in stunted organ growth and produced immune system problems in the rats.

He sent off a paper to one of the most prestigious scientific publications in the world, an English  journal, The Lancet.  It was reviewed and accepted.  That was his first mistake.  The second one was when he was interviewed on BBC national television about his discovery.  The Head of the Rowett Institute called Dr Pusztai and congratulated him on his presentation.

Three days later, the roof fell in.  He was locked out of his laboratory and subsequently fired. His wife and co-author also lost her job at the institute, and the wrath of the scientific establishment came down on his head.  Letters came pouring into The Lancet, criticizing his paper and The Lancet for having accepted it.  They ranged from charges that his controls were inadequate, his interpretation of his data incorrect, to insinuations that he had totally botched the experiment by mistakenly putting an entirely different, toxic chemical into the potatoes.  The Editor of The Lancet, to his credit, vigorously defended the scientific value of Dr Pusztai’s paper.

After weeks  and months of such a bombardment, Dr Pusztai and his wife decided to take a vacation to get away from all the stress.  That was mistake number three, and this is when the story really gets scary.  While he was away, his home got broken into, and guess what was taken – his research data books!  I wonder how much they would bring at a pawn store?  At about the same time his former lab at the Rowett Institute was also broken into.

Perhaps the unkindest cut of all came when rumors were spread that, yes, Dr. Pusztai had been an eminent scientist, but that now he is old and suffers from dementia.  He had become addled.

Back to that evening at Dr. Benbrook’s house on Upper Pack River Road.  Chuck Benbrook runs an internet information service, called Ag BioTech InfoNet.  It is devoted to GE impacts and applications to agriculture, especially pesticides.  Dr Benbrook is an agricultural economist, who formerly worked in Washington D.C. as Executive Director of the Subcommittee of the House Committee on Agriculture .  He met Dr Pusztai at a conference in Paris, and invited him to the US where he had arranged a speaking tour for him.

I spent over three hours dinning and talking with Dr. Pusztai.  I found him to be charming, highly intelligent, and surprisingly unbitter about what had happened to him.  If he is demented, maybe we should all have Alzheimer’s.  He was as sharp as a tack.

 

The Mexican Maze

     University of California at Berkeley (U C Berkeley) graduate student David Quist went down to Oaxaca, Mexico, to show farmers how to test seeds for GMOs.  Oaxaca is known as the birthplace of Corn, and its ancestor plant, Teosinte, still exists there.  It was feared that genes from GM corn (or maize as it is properly known), might, by way of its airborne pollen, get into Teosinte and the form of maize farmed there called Criolla, and turn them into “superweeds” i.e. wild forms of domestic plants, that because they have been genetically transformed, with let’s say built-in pesticide-producing capabilities, can successfully compete with their agricultural relatives and crowd them out.  For this reason, and because transgenic (GE) crops are considered a particular threat to biodiversity, the Mexican government had declared Oaxaca a GMO-free zone.

Quist needed controls to show the farmers what both positive and negative results looked like. For the positives, he brought along store-bought corn from the US, where at least 40 % of the crop is now GMO.  He used native Mexican Criolla for the negatives.  But, something was wrong.  He kept getting positive signals from the Criolla.

Quist took samples of the Criolla back to Berkeley where he and his major professor, Dr Ignacio Chapela of the Department of Environmental Science, decided to do more detailed studies.  They came up with two major findings: (1) Much of the Criolla had a Cauliflower Mosaic Virus(CMV) gene in it.  CMV is used by Molecular Geneticists as a Promoter, typically used to “turn on” or activate inserted foreign genes; and (2) There was other foreign genetic material in these plants, and (3) most importantly, it had moved around in the Criolla DNA.  Genes are not supposed to do this.  They are supposed to sit tight where they are put.  If they move around, they could have different, unexpected effects.

Chapela and Quist submitted their findings to Nature, perhaps the most respected and tough-to-get-into journal in the world.  Their paper underwent four rigorous peer-reviews in eight months, was accepted and published.

 

The Death Star

     The proponents of GMO’s insist that GE is a safe, predictable, and exact science.  They give the impression that they know and can control where each inserted gene goes in the genome, and how it is expressed.  They do not talk much about the possibility that these genes could be passed to other plants.

This paper challenged all of those assumptions, and the reaction was not slow in coming.  Several Letters to the Editor were sent to Nature by both present and former graduate students and others who had connections with the Department of Plant and Microbial Biology, across the campus at U C Berkeley.

Plant and Microbial Biology had recently signed a contract with bioengineering giant, Syngenta, for which they received twenty five million dollars.  In turn, they agreed to do research for Syngenta and to put Syngenta employees on their Board of Directors.  Even in these days of megabucks, this is a lot of money for one department.  Quist and Chapela had been among a lot of people at the University who had opposed the deal, concerned that it would encourage research that favored genetic engineering and curtail  studies that did not.  We shall see how this plays out.

The letters were unusual for a scientific publication.  There were the usual challenges about possible errors in: techniques, controls, statistics, and interpretations.  However, there were in addition,  ad hominem arguments, accusing Quist and Chapela of allowing their political convictions to sway their research conclusions. There were also allegations that they did not have appropriate scientific backgrounds to understand the intricacies of GE.         Nature ran an editorial that for the first time in 133 years of publication, rescinded support for a paper which however they did not ask to be withdrawn.  In addition, in an unusual move, Nature asked Quist and Chapela to retest their samples using a different technique, and gave them a scant four weeks in which to do it.  They actually accomplished this, and confirmed their original results.

AgBioWorld Foundation, a pro-biotech web site run by Tuskegee scientist C.S. Prakash, was a center for criticism of Quist and Chapela.  It posted many emails critical of them, and curiously enough, 60 of the emails seemed to come from two persons, Mary Murphy and Andura Smetacek.  This caught the eye of an enterprising columnist, Jonathan Matthews, from the British publication, The Ecologist, who succeeded in tracing the emails to the Bivings Group, a Washington PR firm. One of Bivings’ largest customers is another bioengineering giant, Monsanto.  Bivings specializes in ‘Internet Advocacy’ campaigns and ‘Viral Marketing’.  In other words, Bivings floods internet postings and chat groups with anonymous or bogus correspondents, in an attempt to influence opinions favorable to their clients.

Matthews discovered that neither Murphy nor Smetacek are real people. He also revealed that AgBioTech was linked to Bivings on the internet.

GMOs have become a multibillion dollar business, very important to the AgBioTech industry and to the governments of the United Kingdom and the U.S., which support these businesses. This industry has many allies in the molecular biology field, whose prestige, research money, and very jobs depend on the public’s perception that GE is a good thing.  These institutions  will go to great lengths to protect their investment, and they will oppose anyone who tends to cast doubt on the worth and safety of their discoveries.  And, they do not always play fair.

An analysis of these circumstances shows a clear pattern of strategy. Attack the dissenters’ science and methodology through letters to the editor in scientific journals, internet web sites, and press releases from scientific organizations, controlled or influenced by the judicious use of industry money.  In this way, divert the argument away from biological conclusions and toward experimental techniques.  Make personal attacks, either upon the investigators integrity or competence, or better yet, both.  Finally, attempt to destroy their careers, thus preventing them from doing further research along these lines, and as a warning to other scientists that research into the safety of GMO’s will not be helpful to their careers.

I will bring you up to date about Drs. Chapela and Pusztai.  Quist and Chapela’s results have been confirmed by several other investigators. Dr. Chapela recently came up for tenure at Berkeley.  He was supported both by his own department and by the unanimous vote of the university tenure committee.  In an unprecedented move, he was denied tenure by the Chancellor.  He will have to leave the University.  Protests have been organized and letters circulated by students and faculty.

As for Arpad Pusztai, veterans of the Hungarian uprising are not creampuffs.  They are survivors.  Dr. Pusztai has started an organization with a web site, devoted to telling about the other, darker side of GE.

 

For more information on GEs and GMOs, we refer you to Dr. Fischman’s first article, “The Dark Side of Genetic Engineering,” which appeared in The Reader, vol. 2, No. 1, Jan. 6. 2005.

 

 

Cancer, Genetically Modified Organisms (GMOs), Molecular Genetics

The Dark Side Of Genetic Engineering

Jun 27th, 11 / 1 Comment

 

 

 

THE DARK SIDE OF GENETIC ENGINEERING

Ken Fischman, Ph.D.

"Everything has both intended & unintended consequences, & the intended

consequences may or may not happen, but the unintended consequences always do."

                                                      Dee Hock, former CEO of VISA International

         In 1988, Showa Denko, a Japanese pharmaceutical company, shipped the first batch of genetically engineered L-Tryptophan to the United States. L-Tryptophan is an amino acid, normally contained in all of our cells. Naturally-derived L-Tryptophan had been sold over the counter for decades to thousands, perhaps millions, of people to relieve symptoms of insomnia or depression. There had never been reports of any ill effects.

         The genetically engineered L-Tryptophan killed 37 Americans, more than 5,000 others came down with a hitherto unheard of disease called Eosinophilia Myalgia Syndrome, and many were permanently injured. 

         Showa Denko's attorney admitted in federal court that it was most likely that the genetic engineering had caused the calamity. Just prior to the trial, Showa Denko destroyed the original batches of bacteria from which the L-Tryptophan had been extracted.  Showa Denko was clearly at fault, but because the bacteria were no longer available for analysis, it could never be definitively proven that it was specifically the genetic engineering that did it. The Food and Drug Administration (FDA) declared that it was not the genetic engineering that was at fault, and the deaths and injuries were probably due to some manufacturing error. Instead of banning only the genetically engineered variety, they banned all over-the-counter sales of L-Tryptophan.

         When Watson and Crick unlocked the secret of DNA in 1956, they fundamentally changed our world. They enabled scientists to understand many of the basic properties of inheritance. This was followed by the introduction of techniques enabling scientists to manipulate those processes in order to alter living organisms in ways that had never before been possible. In rapid succession, scientists deciphered the code found in the sequence of molecules along the long DNA chain, and discovered that DNA produced a similar molecule called RNA, which in turn produced proteins. Some kinds of proteins make up most of our cell structures, while others function as enzymes, controlling essential bodily processes. This new field of science is called Genetic Engineering (GE) and the new forms of life produced by it are termed Genetically Modified Organisms (GMOs). They are brought to you by modern wizards called Molecular Biologists.

         Molecular Biologists have been able to decipher the genetic code laid out in the linear sequence of genes and identify many of their functions.  They can snip them out of the chains of DNA and insert them in the cells of other organisms. Farmers, animal husbandmen, and scientists have been breeding animals and plants for thousands of years in order to produce new combinations of characteristics. However, up until now, these characteristics had always been ones that had preexisted in some members of the same species. Through eons of evolution, living organisms accumulated combinations and sequences of genes that for the most part work together harmoniously. However, the techniques of Molecular Biology shorten the time dimension and leapfrog the species barrier. For example, it is now possible to take the "antifreeze" gene from Flounders, a cold-water species of fish, and insert it into the genome (total array of genes in an organism) of a potato! This enables GE potatoes to survive periods of frost, and to extend their growing seasons. Thus, scientists can now combine genes that had never before been in the same organism.

         The wonderful potentialities of this science have been emphasized for years by molecular biologists, the medical establishment, agribusiness, and government itself. They tell us that they will be able to cure humanity's illnesses, produce wonder drugs grown in genetically-altered animals, grow made-to-order organs for transplantation, feed the starving millions of mankind, etc. However, none of these institutions talk about the dark side. This article explores the dark side.

         In 1992 the FDA issued a ruling, stating that genetically engineered foods are "substantially" like natural foods, and therefore do not need to be regulated.  This has come to be known as the "Substantial Equivalence" rule.  The significance of this ruling was that the food industry would not have to perform safety studies and clinical tests on GMOs, such as are required before new drugs come onto the market. The ruling also removed much of the oversight that the FDA would exercise on drugs after they reached the market. The Federal Government could thus argue, and subsequently did, that because these GE foods are just like regular foods, there is of course no need to label them in order to distinguish them from other, non-GE foods.

         European countries, on the contrary, have adopted a different approach  to the marketing of GMOs.  They have put the onus of proving the safety of these foods on the manufacturers, by invoking what is called the "Precautionary Principle".  This states, in part, that "When an activity raises threats of harm to human health, or the environment, precautionary measures should be taken even if some cause and effect relationships are not fully established scientifically.  In this context, the proponent of an activity, rather than the public, should bear the burden of proof."

         Due mostly to these fundamentally different approaches, the United States and the European Union are locked in a struggle, and the Bush administration has recently appealed to the World Trade Organization to issue substantial fines on European countries which do not allow importation and sale of GMO food from the US.

         It has been repeatedly stated by both government and food industry spokesmen that there have been no documented cases of someone being harmed by GE food.

         In light of these claims, an interesting incident occurred in 1996. Pioneer Hybrid, then the largest seed company in the world, wanted to make an improved soybean. Soybeans lack some of the 21 Essential Amino Acids (EAA) that human beings and most other animals need for life, but cannot produce on their own. Most of us get our EAAs from meat. Vegans, however, must carefully balance the types of plants they eat in order to make sure that they get all 21 EAAs in their diet. Pioneer extracted a gene from Brazil nuts in order to increase the soybean's production of another amino acid, Methionine. They then gene-spliced it into their soybeans in an effort to improve their nutritional value, and hopefully the company's profitability.

         Just before this GE soybean was scheduled to go on the market, it came to the attention of some University of Nebraska scientists. By a stroke of good luck, they just happened to have some blood sera from people who were allergic to Brazil Nuts, and they decided to test these beans on it. They got a strong allergic reaction. Quite a few people are allergic to Brazil nuts, and eating these soybeans might have killed many of them. Obviously, something else besides the gene for the amino acid had been transferred into the soybeans.

         When genes are to be introduced into host cells, they do not come alone. After the donor DNA has been cut into many pieces, it is then inserted into bacterial plasmids (circular bacterial DNA), and in this form, the genes can be duplicated to any number necessary. Then, they must overcome the host cell's defenses against invasion of foreign DNA. This is usually accomplished by attaching a "ferry," – an infectious virus or bacterium – to that gene. The virus or bacterium can penetrate into the cell and insert the gene into the native DNA. A way also has to be found to identify and select those cells in which the new gene has been inserted and to dispose of all cells that do not contain this gene. This is usually done by attaching a so-called Antibiotic Resistance Marker (ARM) gene. This ARM confers antibiotic resistance, usually to Streptomycin. Treating the cells with Streptomycin then kills all cells which do not possess the desired inserted gene.

         Genes do not function all by themselves. Most of them are active during only part of the life of the cell.  They may need the assistance of other genes, called Promoters, which “turn on” or activate them. Therefore, a promoter gene, derived from a virus,  is also attached. These genes may also bring with them uninvited guests. When genes are snipped out of their original DNA chain, the process is not exact. The chain is cut in various places by enzymes, leaving pieces of, or entire neighboring genes, attached to the gene to be inserted. The properties of these DNA Fragments may not be known and their presence may not even be detected.

         From this and other evidence, a reasonable person could draw the conclusion that contrary to what the FDA and food industry say, GMOs used as foods are definitely different from regular foods, and need to be tested and labeled to safeguard the health of both ourselves and the rest of the planet.

         There are several good arguments why GE foods should be labeled. For one, people should have the right to know what is in the food they feed to their families. But even more importantly, if GE foods are not labeled, and something goes wrong, and people get sick and/or die, what could be done to trace the source of the problem? Epidemiologists, those public health officials whose job it is to track down the causes of diseases and other health hazards, would have no way to trace the problem back to the GE foods.

         One more important point. Agribusiness companies such as Monsanto consistently claim that their GE seeds will increase crop yields with these techniques, thus being able to feed the world's ever-increasing human population and avoid famine and starvation. Unfortunately most of the evidence so far demonstrates that on the contrary, most of them either marginally increase or even decrease yields. One theory of why this occurs is that much of the plant's energy has been diverted from normal growth into perpetually producing the inserted gene's product.

         A holy grail of molecular biology has been the hope that GE will one day be able to cure inherited diseases by substituting normal genes for the abnormal ones. For the first government-sanctioned attempt at Gene Therapy, children with a hitherto consistently lethal disease were selected. These so-called bubble babies have non-functioning immune systems, and need to be physically isolated from the environment  in artificial enclosures. They usually die in early childhood from infections against which they have no defense. A number of clinical trials were begun around the world, in which ostensibly normal genes were inserted into such children.  Eleven children were selected for one trial in France. Their physicians were optimistic due to the preliminary results. Most of the children showed improved immune functioning. Then one boy came down with Childhood Leukemia. They assumed that this was an unfortunate coincidence. A few months later a second child developed Childhood Leukemia.

         Analysis of their DNA showed what had happened. In the first child the Promoter gene accompanying the therapeutic gene had landed square in the middle of an Oncogene called LMO-2, and turned it permanently on. An Oncogene is a gene, probably needed for normal development, which if switched on permanently, causes cancer. Analysis of the other child's DNA provoked  much more concern. The same Promoter gene landed near the same Oncogene, but not on it. Promoter genes show a gradation of effects, depending how close they are to the gene in question. The closer, the stronger the effect.  All gene therapy trials were immediately stopped.

         The results of this trial are exceptionally chilling. It showed that it matters very much where in the host genome the foreign gene is inserted. The fact is that the scientists have no idea of where the gene is going to land; where, if any, there is a "good" place to land; and no way exists at this time to direct it to such a place.  There has been much talk about "targeted gene repair" , but so far scientists have been unsuccessful in directing foreign genes to specific sites, and may never be able to do so. 

         Perhaps even more importantly, the question arises as to whether the insertion of the Promoter gene in the Oncogene in one case and near the Oncogene in the other, was a coincidence. Considering the vast amounts of DNA in a cell, and that a human cell is estimated to contain 10 – 30,000 genes, it is very unlikely that this was an accident. Therefore, we are left with the possibility that when foreign genes are inserted into a human cell, their destination may not be random, but directed, but not by us. In these cases, it was directed to an Oncogene, with tragic results.

         The prospect of worldwide distribution of GMO's is particularly troubling because they differ in several crucial respects from pollution by petrochemicals and radioactive substances.  Unlike chemicals, GMOs can replicate themselves, thus producing potentially immense amounts.  They can mutate(change) their genetic constitutions, and therefore, their properties.  Furthermore they can disperse to other environments, either on their own, or by piggybacking on other organisms by becoming integrated into their DNA.  It is quite likely that if  some of them prove to have deleterious effects, it will be impossible to correct the situation, and put them back in Pandora's box.

         In this new world of GE, which we are entering so rapidly, the term caveat emptor (let the buyer beware) takes on a new and ominous meaning.

The Dark Side of GE, 10/29/03

         

Cancer, Genetically Modified Organisms (GMOs), Molecular Genetics, Programs

Newsletters, 2011

Jun 20th, 11 / 0 Comments

Our Newsletters will begin in July, 2011.

With respect to our wild lands,  it will cover topics, such as Hunter Gatherers, Wilderness Survival, and Predator Prey Relationships, with emphasis on the role of wolves in healthy ecosystems. 

The environment takes in an even wider swath, so the Newsletter will also cover Cancer, Climate Change, Peak Oil, and Molecular Genetics, especially Genetically Modified Organisms (GMOs).

Anything that effects the health of the Earth is grist for our mill, and hopefully of interest to you. We will therefore upon occasion, wander farther afield if it seems relevant to your interests, to such topics as primitive skills, wilderness awareness, the evolution of man, and so on.

 

 

Please check the News category for the latest topics of interest

Ancient Pathways Group, Cancer, Hunter Gatherers, Molecular Genetics, Newsletter, The Earth, uncategorized, Wolves

Only God Can Make A Tree

May 4th, 11 / 0 Comments

Only God Can Make A Tree, But Monsanto Might Patent It

By Ken Fischman, Ph.D.
Sandpoint Reader

“Science is too important to be left to politicians or to a scientific establishment increasingly in bed with big business.”
                          – Dr. Mae Wan Ho, Institute for Science in Society (ISIS)

   
    What is the one thing that a Christian fundamentalist and a secular liberal can agree upon? That the U.S. Patent Office (USPO) was dead wrong.  

    Legend has it that the Patent Office thought that they were merely granting a patent for a particular sequence of chemicals, but by approving a patent on an oil digesting bacterium, they had granted the world’s first patent on a living organism.
   The fundamentalist would assert the USPO had committed blasphemy. The Progressive might turn to the Greek myth of Pandora’s Box, saying the USPO had released into the world all the troubles in that box.

     It all began in the 1970s when Dr. Ananda Chakrabarty, a biochemist at General Electric, filed an application with the USPO for a patent on an industrial process for destroying waste oil.

     In 1980, the U.S. Supreme Court upheld the validity of Dr. Chakrabarty’s application by a 5-4 decision. The Patent Office announced that henceforth they would consider “non-naturally occurring, non-human multi-cellular living organisms, including animals, to be patentable subject matter.”  

     The lid was off Pandora’s Box. It yawned wide open.

Genetic Engineering, Cloning and Chimeras:

     In order to understand the controversies emerging from this and similar rulings, we need to examine the scientific techniques used to produce these new kinds of organisms.

     Genetic Engineering (GE), cloning and the production of chimeras (pron. Ki-mee-rahs) are three very different techniques which are often confused in the public’s mind. All involve rearrangement of DNA, the chemical from which genes are made.

     GE operates on the molecular level, and involves cutting out a piece of DNA (the “Trans-gene”), from one organism and its insertion into the genetic material of another organism.  

     For example, the “anti-freeze” gene from a Flounder can be inserted in potatoes, thus conferring on them the ability to survive severe frosts.    

     Chimeras are produced using cells and whole animals. Embryonic cells or tissues from another animal are injected into an embryo, thus producing an animal which is composed of two genetically different kinds of cells.

     In one recent experiment, human embryonic brain cells were injected into a mouse embryo. They migrated to the mouse’s developing brain, thus producing a mouse whose brain contained 1% human brain cells. Scientists are now wondering what would happen if they injected enough human cells to make a mouse whose brain was 50 % or even 100%  human. Care to speculate?  

     Here’s another chimera scenario to contemplate.

     When stem cells are taken from a very early embryo and injected into a different embryo, these cells are capable of developing into any kind of adult cell, depending on the nature of the host cells surrounding them. Suppose human stem cells were introduced into a male mouse embryo, migrated to the mouse’s reproductive glands and developed into sperm.  

     Suppose the same thing was done with a female mouse. Suppose these two mice were born and later mated. Of course, the Disney Company already has a copyright on Mickey Mouse.

     Cloning operates on the cellular level. A cell’s nucleus contains the genetic instructions for the production of an entire animal. In this technique, a nucleus is removed from an adult animal’s cell, then placed inside the egg of another whose nucleus was previously removed.

     The egg is then placed in the uterus of yet another animal and allowed to go to term, thus producing an animal genetically identical to the one from which the original nucleus was taken.

     This is how the now famous “Dolly, the Sheep” was produced.

     Human clones have already been created by transferring the genetic material from human cells into the de-nucleated eggs of cows and pigs. This was done not to create embryos and bring them to term, but in order to provide stem cells.  

     Even many of those who support legalized abortions find it disturbing that human embryos have been deliberately created in order to provide material for transplantation or other uses.  

     Somehow, they sense that it is different from using stem cells from embryos left over from in vitro fertilization procedures – procedures by which barren couples can have children. Those embryos would have been discarded anyway.

     I understand how they feel.

     When I started out in genetic research, I ran a laboratory that analyzed human chromosomes. Most of our work came from physicians who suspected chromosome defects in unborn fetuses. Although I knew that people might opt for abortions on the basis of my analyses, it did not bother my conscience because I was aware that that such defects usually result in gross birth defects.  

     However, with the passage of time, many requests for analyses started to come from people whose names identified them as coming from cultures which did not value women. I put two and two together and realized they were electing to abort only on the basis of gender. I was disturbed at the use of my expertise for such purposes, and this was one reason I switched my research interests.

The Wonderful World of Patent Laws

     Fish that glow in the dark, goats that produce spider silk in their milk, giant Salmon, etc. These are the GE stories that most often reach the popular press. They sound like something out of science fiction, and yet they are fact. GE has opened possibilities of fantastic new combinations of living organisms, never seen in the natural world.

     However lurid and eye-catching these stories are, there lurks beneath their surface even more far-reaching implications. If you really want to scare yourself silly, welcome to the wonderful world of patenting living organisms.      

     Admittedly, patents are not very sexy. If you want a cure for your insomnia, curl up with a good book on patent law some time. However, because GE patents are impacting our lives, you had better learn something about them.

     A patent is a government grant of the exclusive right to make, use or sell an invention, usually for a limited period. Patents are granted for new and useful products made by or thought up by the mind of man.  

     As world trade continues “globalizing,” there are efforts to make the patent laws of most countries compatible. The Trade Related Aspects of Intellectual Property Rights (TRIPS) is an agreement, administered by the World Trade Organization (WTO), for this purpose. Among other things, TRIPS states that all countries must extend their patent laws to include living organisms.

The Legal Morass

     Despite TRIPS, countries still disagree among themselves about the patenting of life forms. The European Union (EU), the collective voice for most of Europe, has issued a directive on patents, part of which states “the human body … including the sequence or partial sequence of a gene, cannot constitute patentable inventions.”

      It also excludes from patenting: human cloning, use of human embryos and modifications of animals, causing substantial suffering without substantial medical benefit.

     The first patent on a human gene was granted by the European Patent Office (EPO) in 1991. The EPO stated at that time that “DNA is not life.” Obviously, the EPO and the EU are not on the same page.

     The Canadian Supreme Court in a 5-4 decision rejected a patent application for a mouse carrying a human cancer gene, saying that that the mouse failed to meet the definition of an invention.

     On the other hand, patent office officials in several countries have expanded the concept of what can be patented, and ruled that living organisms themselves can be patented when they contain foreign genes.

     Another troubling aspect of GE patents is the broadness of their applicability.  

     The Agricitus Company was granted a patent for all GE soybeans. Mycogen was granted an exclusive patent on any insecticide gene in any plant. The broadness of these patents has had serious consequences. Here are a few:

     John Moore was a leukemia patient, who had his spleen removed in 1976. He signed a consent that stated the organ would be destroyed. Without his knowledge, his cells were cultured to produce anti-cancer drugs, now worth billions of dollars. The doctor was listed as the “inventor,” and the patent application was sustained by the California Supreme Court in 1990.    

     Scientists have identified two genes, BRCA1 and BRCA2, which predispose women who to breast cancer.  

     Myriad Genetics has patents on mutations of these cancer genes, and they ordered laboratories to either cease screening for these genes, or pay royalties. Who owns these genes, the women from whom they were taken or a commercial entity?

     A U.S. company, Biocyte, owns a patent on all umbilical cords from fetuses and newborns because they worked out a process for isolating and freezing them. They now have the right to demand fees from anyone extracting and using any human umbilical cells.

     Genetic material is also being taken from Third World countries, being marketed and sold without the consent of the people from whom they were taken.

     For instance, blood samples have been taken from traditional people, such as the Australian aborigines and the Saami of northern Scandinavia, and the genes isolated from them have been patented and marketed commercially.

     Some pharmaceutical and agri-businesses employ Ethnobotanists, people who gather plants from exotic places. They make use of indigenous peoples’ knowledge of the location and medicinal properties of these plants. The companies claim that these materials only have “value” when they have been extracted and marketed commercially.

     In their eyes, the knowledge and use by traditional people is valueless.

     For thousands of years, Third World farmers have lived sustainedly on small, subsistence farms, saving and exchanging seeds. Now, their way of life is being threatened by patents on GE seeds and food.

      Dr. Mae-Wan Ho says (GE) “crops will further destroy livelihood and self-sufficiency through corporate patents on seeds that farmers cannot [legally] re-sow or exchange, and through Terminator seeds that are rendered sterile, breaking the very cycle of renewal and regeneration that is the essence of life.”
   
Problems Inherent in  GE Technique

     Those who defend the value of GE argue that cells and organisms in which genes have been inserted are similar to natural ones, and so there should be no concern over their safety.

     They gloss over the fact that the inserted genes bring with them a great deal of other genetic material. A piece of a virus is attached to the Trans-gene, enabling it to penetrate the host cell’s defenses against foreign DNA and integrate into the cell. An antibiotic resistance gene from a bacterium is also attached to the inserted gene and used as a marker to distinguish which cells have successfully incorporated the Trans-gene. The Trans-gene is activated by attaching a gene from the Cauliflower Mosaic Virus to it.  

     In addition, some unwanted baggage comes along for the ride. Snipping the Trans-gene out of its original DNA is an imprecise process, and often pieces of or entire neighboring genes are included.

     The location and number of copies of genes have profound effects on organisms. Yet, it is inherent in the GE technique that it can’t be controlled where the gene lands in a cell and how many copies of it are included.  

     It is possible that many of the abnormalities now being reported in GE organisms are caused by the technique itself. For example, some pigs containing Human Growth Hormone genes have arthritis, deformities and blindness. Some sheep with this Trans-gene develop Diabetes.

     GE genes for producing commercially and medically important chemicals, such as hormones, antibiotics and blood components, have been incorporated in farm animals.  The animals can also be used as reservoirs for organ transplants.  
     Critics warn that hundreds of different viruses, specific to pigs, could be transferred to humans along with the transplants. This could be dangerous. For example, many scientists suspect that a virus, somehow leaping the species barrier from monkey to man without the benefit of GE, started the AIDS epidemic.

     There are other dangers posed by GE organisms. They have never been part of our food chain, and therefore they may have unanticipated effects on other animals that eat them and plants that absorb their genetic material.

     Trans-genes are deliberately designed to cross species barriers, invade other organisms’ DNA and became part of it. This has already occurred accidentally in creatures that they were never intended to be part of. For example, genes from GE crops have been detected in soil fungi and in the DNA of bacteria and yeasts in the gut of Honeybees.

Ethical Dilemmas:

     It is easier to count the number of Angels that can dance on the head of a pin than to navigate through the mass of conflicting laws, regulations and opinions concerning the desirability and rightness of patenting life.

     For example, Molecular Biologists and the AgBioTech businesses maintain that the human DNA code is patentable because the intellectual effort to discover it raises it from a discovery to an invention.    

     They contend that people deserve the fruits of their intellectual work and that because GE research is expensive – often taking years from discovery to market – they have a legitimate need to protect their large research investment with a patent.        

     Opponents of GE see many problems with patenting living organisms.  

     They argue that GMOs are expropriations from life, and therefore do not qualify as inventions, but only as discoveries. They claim that the terms used to define these technologies have been made deliberately vague in order to cover up our ignorance of biological processes.

     Many religious denominations have also voiced their concerns.

     The Church of Scotland looks at living organisms as part of God’s creation or a product of Nature. It asks how humans can claim to have invented a GE animal or plant just because they have added one or two genes to an organism that already has thousands.

     In their eyes, extending patents to living creatures violates a normal ethical distinction between what is alive and what is not.

     The late Pope John Paul II, speaking for the Roman Catholic Church, strongly denounced the idea of patenting living organisms or their parts, stating, “ … not everything that is technically possible is morally acceptable.”    

Where is the Beef ?

     In addition, other critics of GE ask, “Where are the health benefits that the industry promised the rest of us?”

     Drs. Jeremy Rifkin, of the Foundation on Economic Trends, and Mae Wan Ho, point out that while the industry is constantly hyping the health benefits of the technology, GE has not cured a single individual of a single disease.

     After more than 20 years and billions of taxpayer dollars spent on programs such as the Human Genome Project, all they have to show are patented gene tests.

     It would be ironic if Right to Life fundamentalists and secular liberals found common cause in their opposition to patenting GE organisms. Together, they would be a force to be reckoned with.

 

      “We are incalculably far away from being able to create life de novo … the argument that the bacterium is Chakrabarty¹s handiwork and not nature’s wildly exaggerates human power and displays the same hubris and ignorance of biology that have had such a devastating impact on the ecology of our planet.”
                       – Key Dismukes, National Academy of Sciences, U.S.

Cancer, Chromosomes and Stress, Genetically Modified Organisms (GMOs), Molecular Genetics

The Empire Strikes Back

May 4th, 11 / 0 Comments

                                    “THE EMPIRE STRIKES BACK”

                  How Agribusiness Treats Scientists Who Question GE Safety

                                             by Ken Fischman, Ph.D.

     My Phone was ringing off the hook.  When I breathlessly reached it, I found my neighbor, Dr. Charles Benbrook on the other end.  “I have a house guest who I think you might want to meet” he said.  “It’s Dr. Arpad Pusztai. We are having a get-together tonight at my house.  Do you want to come?”

     Did I want to come?  Is the Pope Catholic? Do cows give milk?  I had been reading about Pusztai for months in preparing for a lecture I was about to give on Genetic Engineering.  The name Arpad Pusztai (pronounced poos-tee) is not exactly a household word, but in some rarified circles he has rock star status.  He lives in Scotland.  What on Earth was he doing in the little town of Sandpoint, Idaho? I had better begin at the beginning.

      Genetic Engineering(GE) is the science of taking genes from one organism and inserting them in the cells of another, thus making novel combinations of genes that never would have appeared in the normal course of Evolution.  e.g. When a gene for producing the pesticide Bt is inserted into corn, every cell in the corn plant becomes a miniature factory.

      Right from the beginning, there has been controversy about the nature of these new combinations, dubbed Genetically Modified Organisms (GMOs) and their possible effects. Proponents of GE, which include Agribusiness, many Molecular Biologists (scientists who do GE), and government agencies, argue that it will bring great benefits and is safe because GMOs are essentially the same as naturally-occurring organisms.  Its opponents disagree, both as to the so-called “normal” nature of GMOs and as to their possible effects.

     The question arises, how can we determine if GMOs, are safe to use as food and to let into our environment? One obvious way is to do scientifically controlled experiments on their safety.  But because of the official government attitude that GMOs are a priori “substantially the same” as natural organisms, relatively little research into that question has been done.

     In 1998, Arpad Pusztai, who worked in the Rowett Institute in Edinborough, Scotland, received the first grant in the United Kingdom to examine the effects of GMO food on animals.  Dr. Pusztai, who fled his native Hungary during the anticommunist uprisings of the 1950s, is a biochemist, who specializes in nutritional studies.  He has written almost 300 scientific papers and has an international reputation.  He was thrilled to get the grant.  He did not know that it was going to destroy his scientific career.

     Dr Pusztai studied rats fed GMO potatoes, in which a gene from the Snowdrop plant was inserted.  That gene produces a Lectin.  That is a chemical that helps protect plants from insect pests.  He thought that it was going to be a straightforward study that would support the conventional scientific wisdom that GMO plants were just like ordinary plants.  He found instead that the presence of the gene resulted in stunted organ growth and produced immune system problems in the rats.

     He sent off a paper to one of the most prestigious scientific publications in the world, an English  journal, The Lancet.  It was reviewed and accepted.  That was his first mistake.  The second one was when he was interviewed on BBC national television about his discovery.  The Head of the Rowett Institute called Dr Pusztai and congratulated him on his presentation.

     Three days later, the roof fell in.  He was locked out of his laboratory and subsequently fired. His wife and co-author also lost her job at the institute, and the wrath of the scientific establishment came down on his head.  Letters came pouring into The Lancet, criticizing his paper and The Lancet for having accepted it.  They ranged from charges that his controls were inadequate, his interpretation of his data incorrect, to insinuations that he had totally botched the experiment by mistakenly putting an entirely different, toxic chemical into the potatoes.  The Editor of The Lancet, to his credit, vigorously defended the scientific value of Dr Pusztai’s paper.

     After weeks  and months of such a bombardment, Dr Pusztai and his wife decided to take a vacation to get away from all the stress.  That was mistake number three, and this is when the story really gets scary.  While he was away, his home got broken into, and guess what was taken – his research data books!  I wonder how much they would bring at a pawn store?  At about the same time his former lab at the Rowett Institute was also broken into.

     Perhaps the unkindest cut of all came when rumors were spread that, yes, Dr. Pusztai had been an eminent scientist, but that now he is old and suffers from dementia.  He had become addled.

     Back to that evening at Dr. Benbrook’s house on Upper Pack River Road.  Chuck Benbrook runs an internet information service, called Ag BioTech InfoNet.  It is devoted to GE impacts and applications to agriculture, especially pesticides.  Dr Benbrook is an agricultural economist, who formerly worked in Washington D.C. as Executive Director of the Subcommittee of the House Committee on Agriculture .  He met Dr Pusztai at a conference in Paris, and invited him to the US where he had arranged a speaking tour for him. 

     I spent over three hours dinning and talking with Dr. Pusztai.  I found him to be charming, highly intelligent, and surprisingly unbitter about what had happened to him.  If he is demented, maybe we should all have Alzheimer’s.  He was as sharp as a tack.

     University of California at Berkeley (U C Berkeley) graduate student David Quist went down to Oaxaca, Mexico, to show farmers how to test seeds for GMOs.  Oaxaca is known as the birthplace of Corn, and its ancestor plant, Teosinte, still exists there.  It was feared that genes from GM corn (or maize as it is properly known), might, by way of its airborne pollen, get into Teosinte and the form of maize farmed there called Criolla, and turn them into “superweeds” i.e. wild forms of domestic plants, that because they have been genetically transformed, with let’s say built-in pesticide-producing capabilities, can successfully compete with their agricultural relatives and crowd them out.  For this reason, and because transgenic (GE) crops are considered a particular threat to biodiversity, the Mexican government had declared Oaxaca a GMO-free zone.

     Quist needed controls to show the farmers what both positive and negative results looked like. For the positives, he brought along store-bought corn from the US, where at least 40 % of the crop is now GMO.  He used native Mexican Criolla for the negatives.  But, something was wrong.  He kept getting positive signals from the Criolla.

     Quist took samples of the Criolla back to Berkeley where he and his major professor, Dr Ignacio Chapela of the Department of Environmental Science, decided to do more detailed studies.  They came up with two major findings: (1) Much of the Criolla had a Cauliflower Mosaic Virus(CMV) gene in it.  CMV is used by Molecular Geneticists as a Promoter, typically used to “turn on” or activate inserted foreign genes; and (2) There was other foreign genetic material in these plants, and (3) most importantly, it had moved around in the Criolla DNA.  Genes are not supposed to do this.  They are supposed to sit tight where they are put.  If they move around, they could have different, unexpected effects.

     Chapela and Quist submitted their findings to Nature, perhaps the most respected and tough-to-get-into journal in the world.  Their paper underwent four rigorous peer-reviews in eight months, was accepted and published.

     The proponents of GMO’s insist that GE is a safe, predictable, and exact science.  They give the impression that they know and can control where each inserted gene goes in the genome, and how it is expressed.  They do not talk much about the possibility that these genes could be passed to other plants.

     This paper challenged all of those assumptions, and the reaction was not slow in coming.  Several Letters to the Editor were sent to Nature by both present and former graduate students and others who had connections with the Department of Plant and Microbial Biology, across the campus at U C Berkeley.

     Plant and Microbial Biology had recently signed a contract with bioengineering giant, Syngenta, for which they received twenty five million dollars.  In turn, they agreed to do research for Syngenta and to put Syngenta employees on their Board of Directors.  Even in these days of megabucks, this is a lot of money for one department.  Quist and Chapela had been among a lot of people at the University who had opposed the deal, concerned that it would encourage research that favored genetic engineering and curtail  studies that did not.  We shall see how this plays out.

     The letters were unusual for a scientific publication.  There were the usual challenges about possible errors in: techniques, controls, statistics, and interpretations.  However, there were in addition,  ad hominem arguments, accusing Quist and Chapela of allowing their political convictions to sway their research conclusions. There were also allegations that they did not have appropriate scientific backgrounds to understand the intricacies of GE.         Nature ran an editorial that for the first time in 133 years of publication, rescinded support for a paper which however they did not ask to be withdrawn.  In addition, in an unusual move, Nature asked Quist and Chapela to retest their samples using a different technique, and gave them a scant four weeks in which to do it.  They actually accomplished this, and confirmed their original results.

     AgBioWorld Foundation, a pro-biotech web site run by Tuskegee scientist C.S. Prakash, was a center for criticism of Quist and Chapela.  It posted many emails critical of them, and curiously enough, 60 of the emails seemed to come from two persons, Mary Murphy and Andura Smetacek.  This caught the eye of an enterprising columnist, Jonathan Matthews, from the British publication, The Ecologist, who succeeded in tracing the emails to the Bivings Group, a Washington PR firm. One of Bivings’ largest customers is another bioengineering giant, Monsanto.  Bivings specializes in ‘Internet Advocacy’ campaigns and ‘Viral Marketing’.  In other words, Bivings floods internet postings and chat groups with anonymous or bogus correspondents, in an attempt to influence opinions favorable to their clients. 

     Matthews discovered that neither Murphy nor Smetacek are real people. He also revealed that AgBioTech was linked to Bivings on the internet.

     GMOs have become a multibillion dollar business, very important to the AgBioTech industry and to the governments of the United Kingdom and the U.S., which support these businesses. This industry has many allies in the molecular biology field, whose prestige, research money, and very jobs depend on the public’s perception that GE is a good thing.  These institutions  will go to great lengths to protect their investment, and they will oppose anyone who tends to cast doubt on the worth and safety of their discoveries.  And, they do not always play fair.

     An analysis of these circumstances shows a clear pattern of strategy. Attack the dissenters’ science and methodology through letters to the editor in scientific journals, internet web sites, and press releases from scientific organizations, controlled or influenced by the judicious use of industry money.  In this way, divert the argument away from biological conclusions and toward experimental techniques.  Make personal attacks, either upon the investigators integrity or competence, or better yet, both.  Finally, attempt to destroy their careers, thus preventing them from doing further research along these lines, and as a warning to other scientists that research into the safety of GMO’s will not be helpful to their careers.

      I will bring you up to date about Drs. Chapela and Pusztai.  Quist and Chapela’s results have been confirmed by several other investigators. Dr. Chapela recently came up for tenure at Berkeley.  He was supported both by his own department and by the unanimous vote of the university tenure committee.  In an unprecedented move, he was denied tenure by the Chancellor.  He will have to leave the University.  Protests have been organized and letters circulated by students and faculty.

     As for Arpad Pusztai, veterans of the Hungarian uprising are not creampuffs.  They are survivors.  Dr. Pusztai has started an organization with a web site, devoted to telling about the other, darker side of GE. 

FOR MORE INFORMATION ON THIS ARTICLE, VISIT THE FOLLOWING                                                                WEB SITES

< freenetpages.co.uk/hp/a.pusztai/>               Dr. Pusztai

< biotech-info.net/ >                                             Dr. Benbrook

< agbioworld.org >                                                Dr. Prakash

< tenurejustice.org >                                             support for Dr. Chapela

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Genetically Modified Organisms (GMOs)

Chromosomes and Stress

Dec 19th, 10 / 0 Comments

 

CHROMOSOMES AND STRESS

 Harlow K. Fischman, Ph.D.

College of Physicians and Surgeons, Departments of Medical Genetics, New York State Psychiatric Institute, 722 West 168th Street, New York, N.Y. 10032, and Genetics and Development, College of Physicians and Surgeons, Columbia University

Dennis D. Kelly, Departments of Behavioral Physiology, New York State Psychiatric Institute, and Psychiatry, College of Physicians and Surgeons, Columbia University

Correspondence to: Dr. Harlow K. Fischman, P.O. Box 6025, Englewood, CO – 80155-6095, Voice Mail (800) 228 – 8193, Mail Box #23422.

We thank Dr. Donald Ross for his statistical analyses and John D. Rainer of the New York State Psychiatric Institute for his helpful ideas and support.  We also thank Dr. Mohammed Osman for his cooperation and support; and Emilia Moralishvili, Dr. Osafradu Opam, and Dr. Ludmilla Skaredoff for their technical assistance. Correspondence should be addressed to H. K. Fischman. Dennis D. Kelly is deceased.

We have previously established that acute psychogenic stress in rats induces genetic damage on both the chromosomal and molecular levels (Fischman, Pero, and Kelly, 1996).  Rats subjected to stress showed increases in both the level of Sister Chromatid exchanges (SCEs) and chromosome aberrations (CAs) in bone marrow cells.  The increases, to differing degrees, in SCEs and CAs induced by the exposure of rats to a variety of stressors, such as cold and warm water swim, white noise, and continuous or intermittent foot-shock, demonstrated that this is a general phenomenon of stress.  These stressors differed from each other both quantitatively  and qualitatively.  The varying experimental paradigms followed, demonstrated that such damage can occur in as short a time as 2 hrs, and endure for at least 25 hrs following exposure to stress.  Furthermore, the detection of stress-induced damage by means of Unscheduled DNA Synthesis extended these observations to the molecular level and to yet another cell type, leucocytes (Fischman, et al., 1996).

A focus on the role of stress in disease has led to the development of the field of Psychoneuroimmunology.  Intensive research in this field in recent years has substantiated that there are physiological and molecular, as well as anatomical connections between the Central Nervous System (CNS), and the endocrine and immune systems (Kropiunigg, 1993; Maier, Watkins, & Fleshner, 1994).  For example, the classical experiments of Riley (1975), demonstrated that exposure to or protection from stress can respectively speed up or slow down development of mammary tumors in mice carrying the Bittner oncogenic virus.  Other research has demonstrated that psychological factors, such as stress, contribute to the predisposition, onset, and course of various illnesses, such as depression, infections, rheumatoid arthritis, coronary heart disease, and cancer in humans, and to herpes simplex, poliomyelitis, Coxsackie B, polyoma, and induction and growth of Walker carcinoma and Ehrlich ascites in animals (Dorian and Garfinkel, 1987; Eysenck, Grossarth-Maticek, and Everitt, 1991).  Our hypothesis is that a parallel or related situation exists with regard to stress and the Genetic system.  In the three experiments to be described in this paper, we continued, on the chromosomal level, a systematic exploration of some aspects of psychogenic stress which may effect this system.  We have developed an In Vivo bone marrow technique for examination of SCEs (Fischman, et al., 1996).  SCEs are microscopically detectable interchanges between the replicated chromatids of metaphase chromosomes that are associated with repair of damaged DNA (Latt, 1979).  Exposure to mutagen/carcinogens results in a dose-dependent elevation of SCEs, and when repair-deficient cells are treated with these agents, large increases in SCEs result (Latt, 1979; Perry and Evans, 1975).  Although the mechanism by which SCEs are produced has not yet been elucidated, detection of elevated SCEs has proved to be an exceptionally sensitive measure of the mutagenic potency of environmental agents in mammalian systems (Tucker and Preston, 1996; Wolff, Rodin, and Cleaver, 1977).

In the experiments to be described, we examined: the effect of  more prolonged periods of stress; the possible role of the endocrine system; and the relationship between stress and chemical mutagens.  (1)  Our original studies focused on very short periods of stress, ranging from 3 – 20 min, those usually considered “acute”.  We determined to expand our findings by examining the effects of more extended periods of stress.  If stress periods are lengthened, is damage continued, enhanced, or decreased? This experiment examines whether repeated exposures to the same stressor results in a gradual decline in the genotoxic properties of  stress, as do other bodily responses to stress, such as adrenal activation, which show adaptation( Bodner, Kelly, Brutus, & Glusman, 1978).  A comparison of the levels of SCEs and of CAs in groups of rats exposed to different durations of stress, also examines whether the genotoxic properties of stress display a different time course of adaptation to chronically stressful conditions than do many other bodily functions affected by stress.  We chose to scrutinize extended stress periods of 3 and 10 days.

(2)  The endocrine system plays an integral role in mediating the effects of the nervous and immune systems (Vollhardt, 1991). In order to explore whether it takes an analogous or identical part with respect to the nervous and genetic systems, we examined whether hormones secreted by glands in the  hypopothalamic-pituitary-adrenal axis have an impact on stress-induced chromosome damage.  This was accomplished by an examination of the effects of foot-shock stress on SCE and CA levels in hypophysectomized(Hypox) rats.

(3)  Physical agents, such as UV and ionizing radiation, and many chemical agents, act as mutagens (Bloom, 1981).  Many mutagens increase SCE and CA levels(Tucker and Preston, 1996).  In light of our demonstration of the SCE- and CA-inducing actions of psychogenic stress, the ubiquity of physical and chemical mutagens in our environment (Bloom, 1981), and of stress in our society (Kiecolt-Glaser & Glaser, 1987), it is appropriate to raise the question as to the possibility of interaction between stress and mutagens.  We examined this prospect by exposing rats to both a chemical mutagen, Mitomycin C (MMC), and foot-shock stress.

MATERIALS AND METHODS

In all experiments, the subjects were male albino Sprague-Dawley rats.  They were individually housed with continuous access to food and water, and maintained on a 14 hr light/10 hr dark cycle, with the exception of the Prolonged Continuous Stress experiment.  Whenever the experimental design permitted, all stress sessions, as well as the time of sacrifice, occurred + or – 1 hr from the midpoint of the light cycle. Two hours following a single exposure, or immediately following the last of a series of stress sessions, each animal was lightly anaesthetized with ether, and a Bromodeoxyuridine (BrdU) pellet (250 mg/Kg B.W.) was implanted subcutaneously on the back of the neck.  Twenty-one hours after BrdU implantation, the rat was injected intraperitoneally with Colcemid (0.6 mg/Kg B.W., i.p.).  Two hours later the rats were sacrificed by guillotine, and hindbone bone marrow preparations were made for analysis of SCEs (Allen, Shuler, & Latt, 1978).  Slides were stained according to the method of Goto, Akematsu, Shimazu, & Sugiyama (1975), for permanent fluorescence plus Giemsa (FPG) SCE preparations.  Fifty well-spread and well-stained, apparently unbroken second division (after BrdU addition) metaphases from each rat were selected for SCE analysis.  Only cells with less than 39 chromosomes were excluded (2n = 42 in R. Rattus).  In experiments in which chromosome aberrations were also to be analyzed, 100 first division metaphases from the same slides were selected.  Slides were coded and scored blind.  SCEs were scored as the mean number of SCEs/cell and chromosome aberrations were scored as the mean number of cells with at least one chromosome break in 100 cells (Fischman, et al., 1996).

Prolonged, Continuous Stress  (PCS) 

Ten rats, 11-12 weeks of age, bred and maintained as previously described, and equated for weight, were assigned to three groups, which were subjected to respectively: No-Stress (NS), n = 4; 72-hr Stress (72S), n = 3; and 240-hr Stress (240S) n = 3.  Throughout the stress condition the subject was exposed to constant dim illumination in the test chamber.  Food and water remained freely available.  The stress paradigm consisted of  alternating 30 min sessions of white noise stress and Conditioned Emotional Response (CER) training.  The initial session was always white noise.  White noise was delivered by two independent audio noise generators over two loud speakers mounted 5-10 cm from the Plexiglas test cage in which the subject was housed.  In turn, this cage was enclosed within a larger ventilated, sound-insulated chamber so as minimize the influence of external stimuli and maximize the efficiency of the speaker system generating the white noise.  Combined white noise output was calibrated at 120 db.  Free field noise intensity measurements were made at the completion of each experiment.  CER sessions consisted of audio warning stimuli which were intermittently paired with brief inescapable footshocks according to the following schedule.  The onset of each minute in the CER session had a 50% probability of triggering a warning stimulus.  The duration of the pure tone warning stimulus was fixed at 40 secs.  The offset of each warning stimulus carried a 50% probability of causing a 500-msec shock (2 mA constant current) to be delivered through the grid floor of the test chamber.  The polarity of the grid bars were “scrambled” at a high speed to prevent intensity-attenuating escape responses within the 500-msec duration.  The long-term probabilities of the CER protocol were as follows:  On the average there were 15 warning stimuli per 30 in CER session.  Hence, at 40-s per stimulus, the average subject spent approximately 10 mins of each CER session in the presence of warning stimuli and 20 mins in safety.  With the probability of shock set at P = 0.5 per warning stimulus, the long-term expectancy of shock was 7.5 per 30-min CER session or 180 per 24 hrs.  Although the actual shock frequency was low, the threat of shock was constant and high, for the subject could not predict which minute would be associated with a warning stimulus, and which warning stimulus would be paired with a terminal shock, except in a probabilistic manner.  However, because no free shocks were programmed to occur unless proceeded by a warning tone, the latter was a predictor of shock and hence acquired the properties of a conditioned emotional stimulus.

Hypophysectomy

Seventeen 58 day old rats were hypophysectomized or sham-operated 9 days prior to the start of the experiment. Their age at sacrifice was 67 days.  Animals were divided into 4 groups: Hypophysectomy-Stress (HS),  n = 5; Hypophysectomy-No Stress (HC), n = 4; Sham-operated-Stress (SS), n = 4; and Sham-operated-No Stress (SC), n = 4.  Stressed animals were subjected to 240 Intermittent Foot-Shocks (IFS).  These were administered  as 2.5 mA, 60 Hz constant current pulses of 1-s duration repeated every 5 s for 20 min.  The non-stressed groups (HC and SC) were handled for 3.5 min.  Slides were prepared and scored for SCEs and CAs in the same manner as in the prolonged continuous stress experiment.

Mutagen/Stress

Twenty rats, bred and maintained as previously described, and equated for weight, were assigned to five equal groups, which were subjected to respectively: No Stress (NS), No Stress plus Saline (NSS), No Stress plus Mitomycin-C (NSMC), Stress plus MMC (SMC), and Stress plus Saline (SS). All animals except the NS group were injected subcutaneously with either 10-8 M (f.c.) of MMC, an amount determined in preliminary experiments to approximately double the SCE level, or an equal volume of saline.  The SMC and SS groups were then subjected to IFS as previously described for the hypophysectomy experiment.  This intensity and duration of foot-shock has been shown to increase the SCE level in male rats (Fischman, et al., 1996).  Two hours after injection, or handling in the case of the NS rats, BrdU pellets were subcutaneously implanted.

RESULTS

Prolonged ,Continuous Stress

Both SCEs and chromosome aberrations were subjected to One-Way Analysis of Covariance (ANCOVA).  For chromosome aberrations, P values were derived from one-tailed t Tests :  Overall ANOVA:  F = 15.2, df = 2,7, P<0.005.  For SCEs, P values were derived from two-tailed t Tests :  Overall ANOVA: F = 20.7, df = 2,7, P<0.005.  72 hr and 240 hr Stress groups showed significant and highly significant increases respectively in both SCEs and chromosome aberrations (see Table 1).

SCEs:   The level for the 72 hr Stress group was significantly higher than for the No-Stress group (P<0.05).  SCEs for the 240 hr Stress group were elevated in a highly significant manner over those of the No-Stress group (P<0.0005).  The level for the 240 hr Stress group was significantly higher than that of the 72 hr Stress group (P<0.01).

Chromosome aberrations:  The level for the 72 hr Stress group was significantly higher than for the No-Stress group (P<0.05).  Aberrations for the 240 hr Stress group were elevated in a highly significant manner over those of the No-Stress group (P<0.001).  The level for the 240 hr Stress group was significantly lower than that of the 72 hr Stress group (P<0.05).

SCEs/CAs:  The correlation was extremely weak ( R = 0.185).

Hypophysectomy

Both SCEs and chromosome aberrations were subjected to One-way Analysis of Covariance (ANCOVA).  P values were derived from two-tailed t Tests.  Overall ANOVA: F = 14.3. df = 3, 28, P<0.0005.

SCEs:   IFS elevated the level in both Stressed Sham-Operated  (P<0.0001) and Stressed Hypoxed rats (P<0.0005), compared with their No-Stress controls.  However, there was no significant difference between the No-Stress, Sham-operated and No Stress, Hypoxed groups (P<0.67) or between the Stressed, Sham-Operated and Stressed, Hypoxed (P<0.550 groups.  Overall, the elevation of SCEs in the combined Stressed groups was highly significant when compared with the combined No-Stress groups (P<0.0001).  There was no significant difference between the combined Sham-Operated groups  and the combined Hypoxed groups (P<0.91).  There was a significant difference between groups that differed in both respects, Hypox, No-Stress compared with Sham-Operated, Stress, (P<0.0005). (see Table 2)

Chromosome aberrations:   These results paralleled those found with respect to SCEs.  IFS elevated the level in both Stressed Sham-Operated (P<0.0001) and Stressed Hypoxed rats (P<0.0001) compared with their No-Stress controls.  There was however, no significant difference between the combined No-Stress, Sham-Operated and No-Stress, Hypoxed groups (P<0.62), or between the combined Stressed, Sham-Operated and Stressed, Hypoxed groups (P<0.054).  Overall, the elevation of chromosome aberrations in the combined Stressed groups was highly significant when compared with the combined No-Stress groups (P<0.0001).  Chromosome aberrations in the Hypoxed, Stressed group were elevated in a highly significant manner over the Sham-Operated, No-Stress group (P<0.0001).  There was no significant difference between the combined Sham-Operated groups and the combined Hypoxed groups (P<0.054).  There was a significant difference between groups that differed in both respects, Hypox, No-Stress compared with Sham-Operated, Stressed (P<0.0005). (see Table 2).

Mutagen/Stress

SCEs were subjected to One-way Analysis of Variance (ANOVA).  P values were derived from one-tailed t Tests.  Overall ANOVA:  F = 8.4, df = 4, 15, P<0.001.  There was no significant difference in SCE level between the Home Cage group (NS) and the No-Stress, Saline-injected group  (P<0.15).  There were significant differences between the Home Cage and both the Stress + Saline and No-Stress, MMC groups, which had higher SCE levels (P<0.01 and P<0.0001 respectively).  There was a highly significant difference between the Home Cage and Stress + MMC group, which had a higher SCE level (P<0.0005).  The Saline-injected group compared with the remainder of the groups, all of which had higher SCE levels, in the following manner:  vs Stress + MMC, a highly significant difference (P<0.005),  vs Stress + Saline and vs No Stress, MMC, no significant difference (both groups, P<0.5).  Stress + Saline was compared with No-Stress, MMC, which although it had a higher SCE level, showed no significant difference (P = 0.72).  The Stress + Saline group was significantly different from Stress + MMC, which had a higher SCE level (P<0.05), and Stress + MMC’s SCE level was significantly higher than that of No Stress, MMC (P<0.05) (see Table 3).

DISCUSSION

In many circumstances the physiological and behavioral effects of acute stress have been shown to have different and even paradoxical effects from those produced by prolonged or chronic stress. (Galinowski, 1993; MacLean, Walton, Wenneberg, Levitsky, Manderino, Waziri, Hillis, & Schneider, 1997 ).  In our original studies, subjects were exposed only to a brief, single occurrence of a stressful situation.  However, unlike initial exposure to stress, repeated exposure to the same stressor is known to result in a progressive decline in certain behavioral responses to stress, such as post-stress analgesia (Bodner, et al., 1980).  Stated alternatively, most stress-activated systems in the body eventually develop tolerance to those environmental demands or situations with which they have become familiar through experience.  Consequently, it would seem important to our line of research and to our cytogenetic system to compare the effects of acute and prolonged exposure to a known SCE-inducing stressor in order to determine whether stress-induced cytogenetic damage also shows adaptation.  A number of investigators have examined the effects of stress on tumor induction (Sklar and Anisman, 1981).  Although it is difficult to draw general conclusions from studies in this field because of the use of different systems, stressors, and measures of tumor growth, it nevertheless appears that acute stress enhances tumor growth while chronic stress inhibits it.  This differential response was observed using a wide variety of stressors, such as restraint, electroconvulsive shock, swimming, and noise.  To add to the complexity of this situation, it is even a matter of debate what constitutes chronic stress.  We extended our original observations to 3 and 10 day periods in order to cover periods of stress usually considered chronic in rats.

The data on SCEs comports well with our original findings.  SCE levels in unstressed animals in the PCS experiments, as well as in the Hypox and Mutagen/Stress

experiments, were consistent with those found over a number of years in our laboratory, and which have demonstrated a remarkable consistency.  In a total of 41 animals, the mean level has been 2.62 +/- 0.06 SEM SCEs/Metaphase.  The increase in SCE level in both the 72 and 240 hr PCS experiments demonstrates that the elevation observed during acute stress (Fischman, et al., 1996) also occurs during these longer stress periods.  In addition, the  SCE level was higher in 240 hr stress than in 72 hr stress, and thus may indicate either a dose response-type reaction, mediated by number of anticipated stresses, or an accumulation of SCEs over time.  SCE persistence depends on many factors: tissue type, nature of the inducing agent, number of times the animal was treated with the agent, duration of  exposure, what part of the cycle the cell was in during exposure, and how many times the cell divided prior to analysis (Tucker, Strout,

Christensen, & Carrano, 1986).  In absolute terms, the 240 hr SCE level of 5.01 SCEs/cell was in the same range as the highest obtained in our original experiments (6.36 SCEs/cell with long intermittent foot-shock).  In relative terms, the 240 hr SCE level was approximately double that of the controls compared with 2 1/2 X in the aforementioned foot-shock experiment. (Fischman, et al., 1996)  The situation with respect to CAs however, is different.  Although their level also increased in both 72- and 240-hr stress,  it was higher in the 72-hr experiment.  Thus, it would appear that adaptation occurred with respect to CAs but not with SCEs.  There is one report that indicates that adaptation to psychogenic stress may protect against the effects of a chemical mutagen.  Meerson and his co-workers adapted mice to moderate periodic hypoxia and repeated electric pain stresses of moderate intensity.  Treatment of unadapted animals with Dioxidine induced CAs in 11% of bone marrow cells, whereas adapted mice apparently had less than half this amount (Meerson, Kulakova, & Saltykova, 1993).  One possible explanation for the disparity between CAs and SCEs in the PCS experiment would be that the mechanisms for the induction of SCEs and CAs, in so far as they are presently understood, are different, especially those aspects which impact their accumulation and duration.  For example, researchers have reported the persistence of SCE levels, days and even weeks subsequent to a single exposure to a chemical mutagen(Tucker, et al., 1986).  The persistence of CAs in rat bone marrow cells may be of shorter duration.  If adaptation occurred sometime during the 240-hr stress, there may have been a loss or repair of chromosomes with breaks although during the same period SCEs persisted.

There may be other factors besides the phenomenon of adaptation, that make PCS different from acute stress with respect to chromosome damage.  Laudenslager and his colleagues showed that Concanavalin A-induced lymphocyte proliferation in rats is suppressed by inescapable, but not escapable shock (Laudenslager, Ryan, Drugen, Hyson, & Maier, 1983). This suggests that the ability of an organism to exert at least partial control over a stressor may be an important parameter of the degree to which stress interacts with cellular processes.  This line of research might be fruitfully pursued in future investigations of the effects of stress on the genetic system by analyzing genotoxic endpoints, using the “learned helplessness” paradigm.

Two obvious differences from our original experiments on acute stress are that the number of stress events have increased and that the time during which the rats are actually being stressed and during which they are under threat of stress has also increased.  Experiments, in which these factors are varied, may further elucidate their roles. The shock intensity in the PCS experiment (2 mA) differed from that used in the Hypox and Mutagen/Stress experiments (2.5 mA).  Thus, the PCS experiment is not comparable to the Hypox and Mutagen/Stress experiments with respect to shock intensity, and we do not know if these two intensities differ in genotoxicity.  A 14 hour light and 10 hour dark cycle was routinely used in the Authors’ laboratory.  The sole exception was that of the PCS experiment, during which the shock chamber was continuously dimly lighted for the duration of the procedure.  It is not possible to definitively state that there was no stress associated with this schedule because the controls in the PCS experiment were not exposed to constant dim illumination.  However, the control animals were essentially comparable to the controls in all other stress experiments performed in our laboratory over a number of years.  This is demonstrated by the consistency of their SCE levels (2.75 SCEs/metaphase).   

            In the search for mechanisms by which psychogenic stress is converted into chromosome and DNA damage, the endocrine system is a prime candidate, mainly due to its role in mediating between the nervous and immune systems. The Hypothalamus (HT) is known to be involved in stress reactions.  Corticotrophin Releasing Factor (CRF), produced in the HT, stimulates the Anterior  Pituitary to produce ACTH, which, in turn, induces the Adrenal Cortex to produce glucocorticoids (GCC).   GCCs may be involved in genotoxic processes, although convincing evidence for this has yet to be elucidated.  In our Hypox experiment, the lack of difference, with respect to genotoxicity, between sham-operated rats and those which had their pituitaries removed, would seem to preclude a role for glucocorticoids in this process. Nevertheless, because there is a possibility that GCCs can be induced without the intervention of ACTH, we have examined the literature for evidence of genotoxicological effects of GCCs.

Veien and Wulf (1980), indicated that GCCs may play a role in chromosome loss in leucocyte cultures from Sarcoidosis patients. It seems at least equally likely that Sarcoidosis itself, a chronic progressive systemic granulomatous reticulosis of unknown etiology, may have brought about the loss.  Sinues et al., in 1992, reported on a prospective study on asthmatic patients receiving glucocorticoids in combination with Theophylline (TP) and beta-adrenergics.  An increase in SCEs was observed.   Most likely, this was caused by the TP for the following reasons: (1) There was no significant difference between the GCC-treated patients and other subgroups, and; (2)  Methylxanthines, the chemical class to which TP belongs, are known inducers of chromosome alterations.  Tedeschi and his colleagues (Tedeschi et al., 1993) showed that recombinant human growth hormone (rhGH) raises the level of SCEs in Bleomycin-treated leucocyte cultures from otherwise normal children receiving therapy for short stature.  rhGH also induced chromosome rearrangements in these cells.  In an interesting paper, Joseph-Lerner et al. (1993), demonstrated that SCE frequency varied with menstrual cycle time, and had a positive correlation with human chorionic gonadotrophin, testosterone and FSH, and estradiol levels.  Finally, Dhillon and Dhillon (1996) showed that Norethisterone, a commonly used, long-acting, injectable oral contraceptive, induces SCEs and micronuclei in vivo in mice and in cultured human leucocytes.

The Autonomic Nervous system has been shown to modulate the effects of stress on the Immune System.  This is most likely accomplished through direct innervation of the major lymphoid organs and through catecholamine receptors on leucocytes, which produce functional changes in immunological cells (Dhillon and Dhillon (1996); Friedman and Irwin, 1997).  Stress-induced genotoxic damage may be brought about through similar or identical pathways.  Hypothalamic CRF, acts on the Sympathetic Nervous System as well as on the HPA axis.  This results in increased levels of corticosteroids, catecholamines, and certain opiates (Friedman and Irwin, 1997).

The results of our Hypox experiment were clarifying, but not definitive.  Both measures of chromosome damage, SCEs and CAs, were elevated by foot-shock stress, as had been observed in our original experiments. Although there was some difference in the level of CAs in hypoxed and sham-operated stressed animals, it did not reach the level of statistical significance.  We conclude therefore, that there was no difference between the Hypox and Sham-operated groups. However, it must be kept in mind that hypophysectomy is an area fraught with many conflicting results. For example, some aspects of stress-induced analgesia are attenuated by hypox while others are enhanced (Kelly, 1982).  The results in the present study do not eliminate the possibility that other parts of the endocrine and neuroendocrine systems may be involved.  Experiments, involving deficits in other hormones, would be appropriate avenues of  further inquiry.  If correlations between such deficits and stress-induced chromosome damage are found, it could be determined whether hormone replacement therapy restores the normal  situation.

In the mutagen/stress experiment, we showed that both foot-shock and MMC individually elevated SCEs. When animals were exposed during approximately the same time period to both the chemical mutagen and to psychogenic stress, the effect on SCE level was the sum of the individually-induced levels.  Thus, it appears that, at least with respect to this particular mutagen, neither interference nor synergism is involved, but instead there is an additive effect.  However, the effects of saline injection complicate the situation.  Although there was no statistically significant difference between the Home Cage controls and the Saline-injected groups, the SCE level induced by saline injection was sufficiently elevated so that there also was no significant difference between the No Stress, Saline and the No Stress, MMC groups or between the Stress, Saline and Stress, MMC groups.  We suspect that it is not the saline itself, but the handling and injection of the animals that acts as yet another genotoxic stress.  Indeed, it was similar observations, in a prior investigation by the senior author, which eventually stimulated this line of inquiry into the genotoxic properties of stress.  During an examination of the long-term effects of heroin on Rhesus monkeys, it was noticed that SCE levels in saline-injected controls, as well as in the heroin-exposed monkeys, had risen significantly (Fischman, Roizin, Moralishvili, Albu, Ross, & Rainer, 1983).  Unlike the heroin-habituated animals, these controls underwent a great deal of stress for 6 months, due to the necessity of restraining them for daily injections.

A logical step in further investigation of genotoxic stress would be to inquire in what ways stress acts like mutagens.  One obvious direction would be to see how much genetic damage stress can cause.  We have been able to induce SCE levels as high as 6.36/cell (Fischman, et al., 1996), an elevation equivalent to those induced in mice by 9.0 X 10-7M Mitomycin C (Allen and Latt, 1976).  The large body of epidemiological data collected on so-called stress-related disorders have indicated that stress plays a role in the pathogenesis of  many diseases.  The importance of such a role is, up to now, conjecture (Dorian & Garfinkel, 1987).  Finding the highest levels of chromosome damage that can be induced by stress in experimental animals would give an estimate of the mutagenic potential of stress, compared to that of known mutagens, and thus, would give a better idea of the relative effects of chemical mutagens and genotoxic stress.

In such an investigation, the question would arise, as to whether more than one aspect of stress plays a role in the damage.  Each putative stressor could be experimentally dissected to examine such aspects as intensity, duration, pattern, adaptation, inescapability, and unpredictability.  Further investigation should attempt to discern whether there are stress equivalents of the dose response effect of chemical mutagen concentration.  There may be a “dose”-related increase in SCE level as some parameters of stress increase.  If such exist, they might be useful in titrating the effects of various types of stressful situations.  Such a phenomenon could be useful in an experimental paradigm, and cellular mechanisms involved in stress-related disorders might be analyzed conveniently in a laboratory animal model.  Two methodological assumptions underlying this approach are: (1) that exposure of the organism to behavioral stress is analogous to the presence of chemical mutagens in terms of the nature of the genotoxic damage induced, although not necessarily in terms of a common mechanism, and (2) that it is appropriate to utilize SCE levels as a “biological dosimeter” of this damage.

There have been a few reports of genotoxic damage in stressed animals.  Bone marrow cells of rats subjected to long-term stress after exposure to Cyclophosphamide, were reported to show an increase in mutagenic effect, with a higher frequency of translocation-like chromosomal interchanges, but not chromosome fragments, in rats from lines with a “high level of excitability” compared with those with “low-excitability” (Bykovskaia, Diuzhikova, Vaydo, Lopatina, & Shvartsman, 1994).  The functional state of the nervous system was measured by the response of the tibial nerve to electrical stimulation, after 15 days of daily stress caused by randomly presented pairing of light and unavoidable electric shock. This is claimed to give a general indication of the level of excitability of other parts of the nervous system (Vaydo, Shiryaeva, and Lopatina, 1993).  In a more recent report, an increase is described in CA rate in “highly excitable” rats when subjected to short-term emotional and analgesic stress. This was described as “low mutagenic” (Diuzhikova, Bykovskaia, Vaydo, Shiriaeva, Lopatina, & Shvartsman,  1996).   Thus, acute and chronic stress would appear to have paradoxical effects in inducing chromosome damage in rats with different thresholds of nervous system excitability.  One is tempted to make an analogy with different human personality types, which have been implicated in disease etiology (Fox, 1988), but in the absence of more solid evidence, such a conclusion is highly problematic.  Another possible interpretation is that the 15 days of stress used in distinguishing types of nervous system excitability, may have adapted the animals  to induction of CAs by that type of stress, but not to their induction by the acute stress, which apparently was not only quantitatively, but qualitatively different.

There are, as yet, few studies directly or tangentially linking stress-induced cytogenetic damage observed in laboratory settings, with similar findings in humans .  There is one intriguing report of increased SCE levels in soldiers deployed to Kuwait during the Iraq war The authors attribute this increase to hydrocarbons produced in oil fires.  However, they also speculate about other causes, such as psychological stress associated with being in harm’s way during a war.  (McDiarmid, Jacobson-Kram, Koloder, Deeter, Lachiver, Scott, Petrucelli, Gustavison, & Putman, 1995).  The SCE increase might also be caused by some combination of stress and mutagen action, such as exposure to N-Mustard gas (New York Times, 1996), similar to the mutagen-stress experiment described in the present study.  Another study reported a significant SCE increase in five volunteers after sleep deprivation (Bamezai and Kumar, 1992).  Sleep deprivation could be a psychological stress.  Morimoto and his colleagues found a higher level of CAs and SCEs in blood cells of people with “poor lifestyles”.  Not having too much perceived stress was included among the criteria for “healthy lifestyles”.  They state that cell cultures from males who have “good lifestyles” do not show as much increase in SCEs when treated with MMC as do cultures from men with “poor lifestyles”.  They report similar results for cultures treated with Ara-C, an inhibitor of radiation damage repair (Morimoto, Takeshita, Take-uchi, Maruyama, Ezoe, Mure, & Inoue, 1993).  In 1996, Silva and his co-workers, reported on SCE analyses of various categories of workers exposed to noise and vibration.  Among the groups studied, were helicopter pilots, and these exhibited high SCE frequencies.  The authors speculated that these results may not have reflected direct action of physical agents on DNA, but rather stress-induced pathophysiological alterations (Silva, Carothers, Branco, Dias, & Boavida, 1996).

Another line of investigation might examine a possible connection between the genotoxic effects of behavioral stress and those of heat-shock, until recently regarded as a phenomenon acting primarily on the physical and chemical level.  Heat-shock and behavioral stress may have a final common path.  It has been well-established that Heat Shock Proteins (HSPs) can be induced in bacteria and cultured mammalian cells subjected to toxic physical or chemical environments.  Psychogenic stresses in intact animals have also been found to induce HSPs in both brain and gut.  Restraint-water immersion stress of rats has been reported to have significantly increased the level of cerebral HSP70 mRNAs, perhaps indicating a protective role for families of HSPs in mammals under pychophysiological stress (Fukudo, Abe, Hongo, Utsumi, & Itoyama, 1995, Fukudo et al., 1997).  In addition, it has been reported that stressed rats which have been adapted, show a greater increase in HSPs than do stressed but unadapted animals (Meerson, Malyshev, and Zamotrinskii, 1993).  Meerson suggests that the antimutagenic effect of stress adaptation is likely to be accounted for by the stabilizing action of HSP (Meerson, Kulakova, & Saltykova, 1993).  However, Vamvacopoulos and his colleagues reported reduction of HSP90 in adapted rats (Vamvacopoulos, Fukuhara, Patchev, & Chrousos, 1993).  Spontaneously hypertensive rats and mice have abnormal HSP70, which is localized in the major histocompatability complex.  In contrast to adapted animals, these animals had low amounts of steady-state HSP70 (Hamet, 1992).  It is possible that the low concentration of HSP70 disposes them to be hypertensive.  Along this line of reasoning, it is interesting to note that CAs produced by 7, 12-dimethylbenz[a]anthracene (DMBA) in the bone marrow cells of hypertensive rats were reported to be three times above that of control rats (Ueda & Kondo, 1984).  A similar situation had previously been described by Pero and his associates, in a study of hypertensive men.  They demonstrated that N-acetoxy-2-acetylaminofluorene (NA-AAF)-induced UDS in lymphocytes showed a linear increase with blood pressure.  Most interestingly, NA-AAF-induced CAs also increased linearly with blood pressure (Pero, Bryngelson, Mitelman, Thulin, & Norden, 1976).

Thus, there is now a line of evidence indicating that a low level, or abnormal type, of HSP makes some animals more vulnerable to genotoxic damage, and linking stress, HSPs, and DNA damage with at least one condition, hypertension.

REFERENCES

Allen, J. W. & Latt, S. A. (1976). In vivo BrdU-332258 Hoechst analysis of DNA replication kinetics and sister chromatid exchange formation in mouse somatic and meiotic cells. Chromosoma, 58(4), 325-340, November 29.

Allen, J. W., Shuler, C. F., & Latt, S. A. (1978). Bromodeoxyuridine tablet methodology for in vivo studies of DNA synthesis. Somatic Cell Genetics, 4(4), 393-405.

Bamezai, R. & Kumar, N. (1992). Sleep deprivation in human males and its effect on SCE rates in chromosomes – a preliminary study. Mutation Research, 283, 229-232.

Bloom, A. D. (1981). Guidelines for cytogenetic studies. In A. D. Bloom (Ed.). Guidelines for Studies of Human Populations Exposed to Mutagenic and Reproductive Hazards. pp. 3-35. White Plains, N.Y., March of Dimes Birth Defects Foundation.

Bodnar, A. G., Kelly, D. D., Brutus, M., & Glusman, M. (1978). Chronic 2-deoxy-D-glucose treatment: adaptation of its analgesic, but not hyperphagic properties. Pharmacology, Biochemistry & Behavior, 9, 763 – 768.

Bykovskaia, N. V., Diuzhikova, N. A., Vaydo, A. I., Lopatina, N. G., & Shvartsman, P. Ia. (1994). Frequency of chromosome aberrations induced by stress and cyclophosphane in bone marrow cells of rats selected for the threshold of nervous system excitability. Genetika, 30(9), 1224 -1228.

Dhillon, V. S. & Dhillon, I. K., (1996). Genotoxicity evaluation of norethisterone  acetate.  Mutation Research, 367(1), 1-10, January.

Diuzhikova, N. A., Bykovskaia, N. V., Vaydo, A. I., Shiriaeva, N. V., Lopatina, N. G., & Shvartsman, P. Ia. (1996). Frequency of chromosome aberrations induced by a single exposure to stress in rats selected for excitability of the nervous system. Genetika, 32(6), 851-853, June.

Dorian, B., & Garfinkel, P. E. (1987). Stress, immunity and illness – a review. Psychological Medicine, 17, 393-407.

Eysenck, H. J., Grossarth, M. R. & Everitt, B. (1991). Personality, stress, smoking, and genetic predisposition as synergistic risk factors for cancer and coronary heart disease. Integrative Physiological and Behavioral science, 26(4), 309-322, October- December.

Fischman, H. K.,  Pero, R., & Kelly, D. D. (1996). Psychogenic stress induces chromosomal and DNA damage. International Journal of Neuroscience, 84, 219 -227.

Fischman, H. K., Roizin, L., Moralishvili, E., Albu, P., Ross, D., & Rainer, J. D. (1983). Clastogenic effects of heroin in pregnant monkeys and their offspring. Mutation Research, 118(1 – 2), 77 -89, July.

Fischman, H. K. & Kelly, D. D., Unpublished.

Fox, B. H. (1988). Psychogenic factors in cancer. In S. Macs, C. D. Spielberger, P. B. Defares, & I. G. Sarason (Eds.). Topics in Health Psychology. pp. 37-55. New York: John Wiley & Sons.

Friedman, E. M. & Irwin, M. R., (1997), Modulation of immune cell function by the autonomic nervous system. Pharmacology and Therapeutics, 74(1), 27-38.

Fukudo, S., Abe, K., Hongo, M., Utsumi, A., & Itoyama, Y. (1995). Psychophysiological stress induces heat shock cognate protein (HSC)70 mRNA in the cerebral cortex and stomach of rats.  Brain Research, 675(1-2), 98-102, March 27.

Fukudo, S., Abe, K., Hongo, M., Utsumi, A., & Itoyama, Y. (1997). Brain-gut induction of heat shock protein (HSP)70 mRNA by psychophysiological stress in rats. Brain Research, 757(1), 146 -148, May 16.

Galinowski, A. (1993). Stress and panic. Immunologic aspects. Encephele, 19 Spec. No, 1, 147-151, March.

Goto, K., Akematsu, T., Shimazu, H., & Sugiyama, T. (1975). Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining. Chromosoma, 53,  223-230.

Hamet, P. (1992). Abnormal hsp70 gene expression: its potential key role in metabolic defects in hypertension. Clinical and Experimental Pharmacology and Physiology Supplement, 20, 53 -59.

Joeph-Lerner, N., Fejgin, M., Ben-Nun, I., Legum, C., & Amiel, A., (1993), The correlation between the frequency of sister-chromatid exchange and human reproductive hormones. Mutation Research, 300(3-4), 247-252, August.

Kelly, D. D. (1982). The role of endorphins in stress-induced analgesia. In K. Vereby (Ed.) Opioids in mental Illness: Theories, clinical observations and treatment possibilities (pp. 260-270), Vol. 398, Annals of the New York Academy of Sciences,  New York: New York Academy of Sciences.

Kiecolt-Glaser, J. K. & Glaser, R. (1987). Psychosocial moderators of immune function. Annals of Behavioral Medicine, (2), 16-20.

Kropiunigg, U. (1993). Basics in psychoneuroimmunology. Annals of Medicine, 25(5), 473-479, October.

Latt, S. A. (1979). Sister chromatid exchanges. Genetics, 92 (1 Pt. 1 Suppl.), 89-95, May.

Laudenslager, M. L., Ryan, S. M., Drugan, R. C., Hyson, R.L., and Maier, S. F. (1983). Coping and immunosuppression: inescapable but not escapable shock suppresses lymphocyte proliferation. Science, 221, 568-570.

MacLean, C. R., Walton, K. G., Wenneberg, S. R., Levitsky, D. K., Manderino, J. P., Waziri, R., Hillis, S. L., and Schneider, R. H. (1997). Effects of the Transcendental Meditation program on adaptive mechanisms: changes in hormone levels and responses to stress after 4 months of practice.  Psychoneuroendocrinology, 22(4), 277-295, May.

Maier, S. F., Watkins, L. R., & Fleshner, M. (1994). Psychoneuroimmunology: the interphase between behavior, brain, and immunity. American Psychologist, 49(12), 1004 -1017.

McDiarmid, M. A., Jacobson-Kram, D., Koloder, K., Deeter, D. P., Lachiver, R. M., Scott, B. G., Petrucelli, B. P., Gustavison, D., & Putman, D. (1995). Increased frequencies of sister chromatid exchange in soldiers deployed to Kuwait. Mutagenesis, 10(3), 263-265, May.

Meerson, F. Z., Kulakova, A. V., & Saltykova, V. A. (1993). The antimutagenic effect of adaptation to stress. Biulleten Eksperimentalnoi Biologii I Meditsiny, 116 (9), 292 – 295, September.

Meerson, F. Z., Malyshev, I. Iu., & Zamotrinskii, A. V. (1993). The generalized accumulation of stress proteins during body adaptation to stress exposures. Biulleten Eksperimentalnoi Biologii I Meditsiny, 116(9), 231-233, September.

Morimoto, K., Takeshita, T., Take-uchi, T., Maruyama, S., Ezoe, S., Mure, K., & Inoue, C. (1993). Chromosome alterations in peripheral lymphocytes as indices of lifestyle and genotoxicity. International Archives of Occupational and Environmental Health. 65 (I, Suppl.), 537-541.

New York Times (1996). Panel says chemical arms might be cause of gulf illness, November 21.

Pero, R. W., Bryngelsson, C., Mitelman, F., Thulin, T. & Norden, A. (1976). High blood pressure related to carcinogen-induced unscheduled DNA synthesis, DNA carcinogen binding, and chromosomal aberrations in human lymphocytes. Proceedings of the National Academy of Sciences, U.S.A., 73(7),  2496-2500, July.

Perry, P. & Evans, H. J. (1975). Cytological detection of mutagen- carcinogen exposure by sister chromatid exchange.  Nature, 258(5531), 121-125, November 13.

Riley, V. (1981). Psychoneuroendocrine influences on immunocompetence and neoplasia. Science, 212, 1100-1109.

Silva, M. J., Carothers, A., Branco, N. C., Dias, A., & Boavida, M. G. (1996). Sister chromatid exchange analysis in workers exposed to noise and vibration. Mutation Research, 369(1 – 2), 113-121, July 10.

Sinues, B., Broto, A., Suarez, M. A., Duce, F., Martinez-Berganza, A., & Bernal, M. L., (1992), Cytogenetic study in peripheral blood lymphocytes from asthmatic patients receiving continued therapy with theophylline. Mutation Research, 280(4), 271-277, October.

Sklar, L. S. & Anisman, H. (1981). Stress and cancer. Psychological Bulletin, 89(3), 369-406, May.

Tedeschi, B., Spadoni, G. L., Sanna, M. L., Vernole, P., Caporossi, D., Cianfarani, S., Nicoletti, B., & Boscherini, B., (1993), Increased  chromosome fragility in lymphocytes of short normal children treated with recombinant human growth hormone. Human Genetics, 91(5), 459-463, June.

Tucker, J. D., & Preston, R. J. (1996). Chromosome aberrations, micronuclei, aneuploidy, sister chromatid exchanges, and cancer risk assessment. Mutation Research, 365(1 – 3), 147-159, September.

Tucker, J. D., Strout, C. L., Christensen, M. L., & Carrano, A. V. (1986). Sister chromatid exchange induction and persistence in peripheral blood and spleen lymphocytes of mice treated with ethyl nitrosourea. Environmental Mutagenesis 8, 345-355.

Ueda, N. & Kondo, M. (1984). Chromosome aberrations induced by 7,12-dimethylbenz[a]-anthrocene in bone marrow cells of spontaneously hypertensive rats (SHR) and control Wistar Kyoto (WKY) rats: time course and site specificity. Journal of the National Cancer Institute, 73(2), 525-530, August 18.

Vamvakopoulos, N. C., Fukuhara, K., Patchev, V., & Chrousos, G. P. (1993). Effect of single and repeated immobilization stress on the heat shock protein 70/90 system of the rat: glucocorticoid-independent, reversible reduction of HSP90 in the liver and spleen. Neuroendocrinology, 57(6), 1057-1065, June.

Vaydo, A. I., Shiryaeva, N. V., & Lopatina, N. G. (1993). Divergent responses to long-term stressors in rat lines selected for different functional states of the nervous system. Behavior Genetics, 23(5), 499-503, September.

Veien, N. K. & Wulf, H. C. (1980), Chromosome analysis in patients with sarcoidosis. Archives of Dermatological Research, 268(1), 9-13.

Vollhardt, L. T. (1991). Psychoneuroimmunology: a literature review. American Journal of Orthopsychiatry, 61(1), 35-47, January.

Wolff, S., Rodin, B., & Cleaver, J. E. (1977). Sister chromatid exchanges induced by mutagenic carcinogens in normal and xeroderma pigmentosum cells. Nature, 265(5592), 347-349, January 27.

Chromosomes and Stress, Molecular Genetics