Wednesday, October 19, 2011

Assessing low doses of radiation: background fact sheet

The discovery of x-ray induced mutations by H. J. Muller in 1927 led to the field of radiation genetics.

By 1944 the following was known:
1. X-rays induce gene mutations that are indistinguishable from those arising by natural means. Muller noted that in his 1927 X-linked lethal detection stock (ClB) that he also obtained X-linked visible mutations and he mapped them to the same locations as their previously found spontaneous alleles from 1910-1926.
2. X-rays induced a class of mutation not previously described, called “dominant lethals”. It was not known if these were point mutations like the recessive X-linked lethals until 1944 when G. Pontecorvo, using a stock designed by H. J. Muller, detected losses of chromosomes caused by aneucentric chromosomes. These were shown to arise from a single break in a chromosome that led to reunions of replicated chromosome fragments forming a potential acentric chromosome and a potential dicentric chromosome. McClintock had independently found such losses using maize and called this the breakage-fusion-bridge cycle.
3. X-rays induce a large number of chromosome breaks that lead to chromosome rearrangements (worked out for radiation induced chromosome rearrangements in the 1930s). This included inversions and translocations which could be detected genetically and confirmed cytologically.
4. In general the induction of X-linked lethals is linear for doses of 100-6000 R.
5. In general the induction of translocations (as a measure of 2 breaks) was to the 3/2 power instead of the expected square. Muller believed this was associated with the practice of x-raying mature sperm (spermatids or spermatozoa) which tend to have most of the nuceloplasm removed from the nucleus and the chromosomes are pressed close to one another and they are highly coiled and compact. This leads to a single path of radiation producing more than one break. This happens less frequently when immature stages are used where the chromosomes are not compacted and when that is done the incidence of breakage approaches the square of the dose.
6. Attenuated doses (studied by SP Ray-Chaudhury at Edinburgh in 1939 for 400 R given in 30 minutes produced the same percent of x-linked lethals as 400 R given over 28 days). Muller argued that this meant individuals exposed to chest x-rays and practitioners not protecting themselves with shielding were at risk to radiation induced damage at low dose. Because 400 R given over 28 days is like getting a chest x-ray every minute [about 0.01R or 0.0017 R per second] . Most chest x-rays take about 1 second to administer. Thus a low dose of 0.0017R per second over 28 days yields 400 R over 28 days. The effects are cumulative and, as Ray-Chaudhury showed, the percent of induced mutations is the same as that received the acute dose.

Between 1944-1960 the following additional findings were added:

1. The breakage-fusion-bridge cycle was the likely cause of radiation sickness among the survivors of the blast effects of the atomic bombs in Japan. The tissues affected and their symptoms (blistering and reddening of the skin, pinpoint ulceration of the intestinal tract, petechiae or rashes of capillary bleeding inside tissue and in the skin, reduced red blood cell count, reduced white blood count, loss of hair, bleeding from the intestinal tract and vagina were all consistent with those tissues (skin, gut epithelium, capillary endothelial cells, and bone marrow) that had frequent cell cycles replacing tissues exposed to mechanical damage (skin gut and blood vessel lining) or that had high turnover (red and white blood cells). Long term survivors could expect cataracts and increased risk to cancers (usually 7 years for the leukemia to appear and about 25 years for the solid tumors to appear)
2. X-rays cause direct hits to genes causing point mutations and they cause conversion of water to peroxides that act as chemical mutagens and alter nitrogenous bases. Stone, Wyss, and Haas in 1949 showed that bacteria directly exposed to radiation gave only slight more mutations than unexposed bacteria placed on x-rayed food in petri dishes. This showed most of the bacterial mutations from radiation are peroxide induced. This would make a substantial amount of mutations induced by x-rays in humans as similar to effects of exposure to peroxides. Humans have about 70% water in their tissues.
3. Charlotte Auerbach in 1945 published her war-time results (classified as secret until the war ended) on chemical mutagens in fruit flies. She used mustard gas and found most of the mutations induced were fractional or mosaic. It was not until the double helix structure of DNA was worked out in 1953 that this was resolved. Fractional mutations receive one DNA strand with a chemically induced lesion and the complementary strand had the normal complementary base. These two lines of cells are present after fertilization—one leading to mutant expression and one leading to normal expression. In fruit flies the chances of such a mosaic having a gonad with mutant cells present is about 20%. Those progeny show full expression of the mutant.
4. The Watson-Crick double helix model of 1953 led to the study of chemical mutagens on viruses. Three categories were identified by Cricks laboratory and by Benzer’s laboratory in the early 1960s. Transitions were chemical alterations of one purine for another purine or of one pyrimidine for another pyrimidine. Transversions were mutant events in which an altered purine led to altered pyrimidine or the reverse. Frame shift mutations were associated with chemical mutagens that intercalated or inserted a ring shaped molecule, jamming the DNA and during replication leading to losses or gains of one or more pairs of nucleotides. Agents like nitrous acid, hydrogen peroxide, and formaldehyde produce chiefly transitions and transversions. Agents like proflavine or quinacrine (multi-ring compounds) acted as frame-shift agents.
5. In 1952 radioactive fallout was first reported by Japanese fishermen who were exposed to it during the US first H-bomb tests in the Pacific. They had severe radiation sickness.
6. In 1954-1963 there were numerous tests of radioactive fallout from USSR and US atmospheric testing. Barry Commoner studied baby teeth and showed that radioactive elements (especially strontium 90) from these weapons testing was falling on grasslands in the US and entering the milk supply of these children in the United States. The teeth would expose photographic film when these were allowed to rest on it for a day or more.
7. The sequencing of DNA became possible in the 1980s and by the year 2000 it could be extended to whole genomes. Any mutation can be identified (they are sometimes called SNPs or single nucleotide polymorphisms) and this opens up a possible way to look at Japanese atomic bomb survivors or their children to see what SNPs have been induced to control matched populations of nearby Japanese cities not exposed.
8. Evelyn Witkin discovered there are repair enzymes in cells. These can repair single stranded breaks, double stranded breaks (the most common from x-ray direct hits), thymine dimers (the type formed by exposure to ultraviolet light), and mismatched pairing. When double strand breaks occur and are repaired it is possible for the wrong bases to be inserted in the patched region. These would be expressed as gene or point mutations. There are human genetic disorders with defective repair enzymes. Such disorders (Louis-Barr syndrome, Bloom syndrome) are characterized by frequent cancers and an early death. Bacteria lacking catalase (an enzyme that inactivates hydrogen peroxide) have higher spontaneous mutation frequencies and higher induced radiation mutations.

Concerns about experimental design

Any geneticist doing experiments on dose-response effects has to know this very complex background to designing a low dose experiment and to guard against errors that would obscure what is being tested or the interpretation of the results. Here are some of the difficulties of doing low dose experiments:
1. In fruit flies it is difficult to do experiments that are below 100 R because the scale has to be very large. In general the lower the dose the larger the scale of exposed and control populations are needed. In a fruit fly mutagenesis experiment this is usually done by examining vials of flies that descend from the F2 of P1 exposed flies. If the P1 is a normal male and the F1 is a heterozygous fly containing a marked chromosome and the exposed sperm, each F1 vial set up tests one sperm from the exposed male. If the control rate is about 1 X-linked lethal per 2000 flies examined then the control group of several thousand vials has to differ from the low dose exposed group of several thousand vials being tested. For a control rate of 1 in 2000 the statistical expectation range is 1-12 out of a test using 2000 vials for the control. Thus if a low dose of 50 R is done for an experiment involving 2000 vials for the exposed there may be an incidence of 13. This has a range of 6-18. Since the lower range of the induced overlaps the higher range of the spontaneous range, these are not significant. A tray of vials may contain 100 vials and this mean 20 trays of controls and 20 trays of experimental. A more likely experiment anticipating these low number statistical fluctuations would have to use more vials, say 4000 controls and 4000 vials of the experimental or a total of 80 trays so that the two ranges do not overlap. That’s a lot of work. Before 1957 grant support was mostly private. Sputnik changed that to NSF and NIH money on a large scale. This is one reason why there were so few large scale experiments testing low doses before the Cold War. The USSR did not have atomic weapons until 1952 so the issue of fall out and radiation protection was not as elevated. For this reason it could not have been a fall-out issue that motivated Muller. Whatever Muller’s views were on radiation safety they long predated the World War II use of atomic weapons. He was raising concerns about radiation safety as early as 1928 when he gave a talk on x-ray induced mutations at Waco, Texas and cautioned physicians that they should protect themselves and their patients by using lead shielding. Bentley Glass, who attended that lecture, said physicians walked out in protest.
The issue of hormesis

Hormesis was introduced in 1888 by Hugo Schultz. It asserts that low doses of toxic agents may have beneficial effects by boosting an immune response (or some other beneficial mechanism in plants and organisms lacking an immune system) to the agent. Edward Calabrese is a major supporter of hormesis in a wide range of agents, including carcinogens, mutagens, pharmaceutical agents, and radiations. He has extended hormesis to a study of low dose linear curves for toxic agents and radiations and he has extended hormesis to the presence or absence of threshold doses for radiation. Calabrese is not a geneticist. He received his PhD in 1973 for a study of metabolism in black blow flies from the entomology department at UMass Amherst. The next year in 1974 he received a D.Ed. degree with a dissertation on logo therapy. Logo therapy is the psychiatric system worked out by Viktor Frankl using meaning and purpose to achieve mental health particularly in stressful situations (Frankl survived the Holocaust concentration camps).
Calabrese through a press release accused Muller of being a liar and a bully in getting federal and international agencies to accept the thesis that there is no threshold for the linearity of radiation dose-effect curve (which presently runs from about 100 R to 12,000 R for fruit flies). Muller relied on the work of Ray-Chaudhury for the attenuated doses as a reason to exclude a threshold dose and to uphold the extrapolation of the linearity curve. An attempt by Curt Stern to use 50 R was done with Ernest Caspari and a letter and manuscript draft was sent to Muller shortly before he left for Stockholm to accept his Nobel prize in 1946. Calabrese claims that Muller was upset over the apparent below expected response to the 50 R exposure. This to Calabrese is deceit despite the fact that most scientists do not cite unpublished work and despite the fact that Muller wrote to Stern some of his first impression objections to the interpretation of the results. When the paper was published both Stern and Caspari acknowledged a low probability (about 1%) of the results being consistent with the linearity hypothesis and they discussed what problems were involved in their experimental design.
It is disappointing that Calabrese did not treat this as a dispute in science (they are common). Instead he chose to make a conspiratorial case, attributing “higher causes” such as radiation protection, ideology, or concern over fallout from nuclear weapons arms races as the basis for Muller’s “repression” of the Stern-Caspari draft although some of those concerns did not arise until 6 years after Muller’s Nobel Prize. He also makes it seem as if all the other work of other laboratories on radiation genetics and the linearity curve should collapse from this one finding under dispute. It is also disappointing that Calabrese offers no molecular mechanisms like those molecular biologists have offered for the induction of mutations to account for this alleged beneficial effect of low doses of radiation or other agents. He ignores the many variables cited in this account and demands an ideal experiment at low doses without complications despite the numerous variables involved in designing such an experiment. He ignores the attenuated doses studied by Ray-Chaudhury and others and does not explain why they accumulated in Ray-Chaudhury’s work if that total dose was spread at less than chest x-ray exposure each minute.

Elof Axel Carlson
Distinguished Teaching Professor emeritus
Department of Biochemistry and Cell Biology
Stony Brook University
And Visiting Scholar, Institute for Advanced Study
Indiana University (Bloomington).
October 18, 2011

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