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Becoming a Scientist |
I was born on December 16, 1928 in New York City. My father,
Maurice U. Ames, who was born in 1900 in New York City, had a J.D. in
law and an M.A. in chemistry. He had originally planned to practice
law, but when the depression hit, he became a high school chemistry
teacher, which he viewed as a less risky occupation. He later became a
high school principal, then supervisor of science for the New York City
public school system, and eventually Assistant Superintendent of
Schools. My father was very smart, not eager to try new things, and
quite placid by nature. My mother, Dorothy Andres Ames, had come to New
York as a young child from Poland. My mother had a tremendous
joie de vivre, and I think I inherited my ebullience and
creativity from her. Despite their very different personalities my
parents got on marvelously well. Someone once remarked to my mother
that my father "has such a wonderful disposition." My mother
replied, "Oh he doesn't have a wonderful disposition. He has no disposition."
I grew up in the Washington Heights area of Manhattan. Every summer
during my childhood I went with my family to Warrensburg in the
Adirondack Mountains, where my father and a group of other New York
City school teachers rented houses on Echo Lake. Those were wonderful
summers. I would collect and study most of the creatures that existed
in the woods around the lake, though my mother never was too
enthusiastic about the mice and the snakes. Every week I would go and
get another stack of books to read from the town library. I was always
curious about the world and loved digging into still another subject,
and my reading interests have remained quite eclectic to this day. I
attended the Bronx High School of Science where I became immersed in
biology and chemistry. I did my first scientific experiments there; I
grew tomato root tips in culture to determine the effect of plant
hormones on their growth. The picture of those white roots growing on
their own when stimulated by hormones stays in my mind. The pleasures
of doing those experiments set me on the path to becoming a scientist.
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Cornell |
I attended Cornell University from 1946 to 1950 and received
my B.A. degree with a major in chemistry/biochemistry. I never was a
top student, either in high school or college. I had only a so-so
memory and was easily distracted by some new enthusiasm (reading all of
Tolstoy or mastering some new folk dance) when I should have been
studying for an exam. Taking required courses was not a thing I could
get very excited about (I am too undisciplined and driven by my own
enthusiasms) though I did well in those few that sparked my interest.
Two such courses from my Cornell days stay in my memory. One was a
history class taught by Professor Marcham in which we investigated
historical incidents by reading all of the original documents, which of
course were quite contradictory, and then tried to determine what was
the most likely reality. The other was a course in biochemical genetics
taught by Adrian Srb. I had already taken several genetics courses, as
I was interested in the subject, and Srb's course got me all excited
because of my background in biochemistry. I applied to various graduate
schools as I was finishing up but was somewhat apprehensive because of my less than stellar grades. I was, in fact, turned down by Wisconsin, but luckily I was accepted by Cal Tech, perhaps because Srb, or one of
my other references, saw potential in me.
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Cal Tech |
I arrived at Cal Tech in August 1950. I chose Professor
Herschel K. Mitchell, a former postdoctoral fellow of George Beadle, as
a mentor and was doing experiments within a few weeks of arriving. Finally I was in my element, lots of research and relatively few courses. Beadle was chairman of the biology department; he and Tatum
had previously pioneered the use of biochemical genetic techniques in
the mold Neurospora, which led to their Nobel prize.
I studied the biosynthesis of histidine in Neurospora, using
mutant histidine-requiring strains involving at least 4 different genes
that Mitchell had isolated. A few months after I got to Cal Tech, I
adapted a sensitive reagent for the imidazole ring, which is the
heterocyclic ring in histidine, to be used as a spray reagent for paper
chromatograms (1). This was the key to elucidating the biosynthetic
pathway. I grew a culture of each mutant strain in low histidine medium
and chromatographed the supernatant. There were about six different
imidazoles in the collection of mutants, though each defective gene had
a unique imidazole set, and one had none (2). My next task was to
identify these imidazole intermediates. I had one very lucky break.
While searching the literature on imidazoles I came across an old
German paper that reported cooking up glucose, ammonia, and
formaldehyde to form an imidazole with a side chain of four carbons,
each containing a hydroxyl group. My intuition told me this was the
solution to my problem. I then cooked up ribose, ammonia, and
formaldehyde, chromatographed the mixture, and found an
imidazole-containing compound with the same mobility and properties as
one of the compounds accumulated by one of the histidine-requiring
mutants. I guessed, and soon showed, that it was imidazole glycerol
(2).
The rest of my thesis went very quickly. While I was doing all of this,
Bernie Davis' laboratory published a paper showing that histidinol was
a precursor of histidine. That turned out to be another of our
imidazoles. A pathway of imidazole glycerol, imidazole acetol,
histidinol, and finally histidine seemed to make biochemical sense. We
also made the double mutants from all of the genes that were involved,
and I determined which imidazole intermediate accumulated. Using this
trick I was able to order the steps in the pathway and found that this
fit with the biochemistry (3). I made the same compounds from the
various pentoses and showed that the substance from ribose, the
D-erythro isomer, was the right one as it was
the same as the accumulated compound from the mutant (4). I soon found
that some minor, slow moving, imidazole-containing spots on the
chromatograms were the true intermediates; these were the phosphate
esters of the compounds. I synthesized imidazole glycerol phosphate by
cooking up ammonia, formaldehyde, and ribose 5-phosphate. I suggested
that the first step of the pathway involved ribose phosphate. Later at
NIH Bob Martin and I showed that phosphoribosyl pyrophosphate was in
fact the precursor of imidazole glycerol phosphate and that it
condensed with ATP, which donated the nitrogen and carbon of the
imidazole ring (5). I worked out a good part of the pathway of
histidine biosynthesis and finished most of the work for my doctoral
thesis during my first year at Cal Tech.
Cal Tech was an exciting place for a budding scientist. I became part
of the group revolving around Max and Manny Delbruck, who had a salon
of sorts, which included play readings, dinners, musicals (I played the
alto recorder, though not very well), and camping trips into the
desert. While at Cal Tech I took both summers off; one summer I took
C. B. van Niel's bacterial physiology course at Stanford
University's Hopkins Marine Station in Pacific Grove, CA and the next
the physiology course at Woods Hole, MA. Both laboratory courses were
extraordinary and expanded my interests. I completed my Ph.D. degree in
June 1953 at the age of 24. I had arrived at Cal Tech as a very green,
very young researcher who was very uncertain that my memory and focus
were good enough to make it in the competitive world of science. I left
with at least some conviction that my curiosity and creativity might
carry me through and that with a little luck I might make it.
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NIH |
I knew I needed to learn enzymology, so after completing my
Ph.D. degree, I took a postdoctoral position in September 1953 in
Bernard Horecker's laboratory at the National Institutes of Health
(NIH). At NIH I fished out the enzymes of the histidine pathway using
the intermediates I had isolated and synthesized at Cal Tech. I had
switched from Neurospora to Salmonella
typhimurium as a result of a collaboration with Phil Hartman at
Johns Hopkins, who was studying the genetics of the histidine mutants
of Salmonella. In 1954, as an independent investigator at
NIH, I began work on gene regulation in histidine biosynthesis using
Salmonella. We showed that the histidine genes, which were
in a cluster in Salmonella, could be overexpressed if
histidine availability limited the growth rate; we also showed that the
enzymes were controlled as a group, "coordinate repression" (6). We
became interested in a mutant found by Hartman, which had a short
region at one end of the cluster deleted, but turned off the function
of all of the intact histidine genes. We concluded that the cluster of
genes was controlled together as a unit by a regulatory sequence.
NIH was a wonderful place to do science. There was enough money for
research and no teaching or committee duties. I interacted with
outstanding scientists and formed many lasting friendships: Gordon
Tomkins, Earl and Terry Stadtman, Maxine Singer, Ira Pastan, Herb and
Celia Tabor, Leon Heppel, David Davies, Marty Gellert, Gary Felsenfeld,
and many more.
I married Giovanna Ferro-Luzzi in 1960. She had come from Rome to do
postdoctoral work at Johns Hopkins University in 1958, and I met her at
the Baltimore-Washington Enzyme Club. We are still remarkably happily
married some 40 years later. When she finished at Hopkins, Gordon
Tomkins gave her a position in his laboratory.
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Cambridge/Paris |
In l961, I took a year of sabbatical leave from NIH. Giovanna
and I divided our time between Francis Crick's laboratory in Cambridge
and Francois Jacob's laboratory at the Pasteur Institute in Paris. It
was a honeymoon year, both personally and intellectually. This was an
exciting time in Cambridge with Crick, Brenner, Perutz, Kendrew, and
innumerable distinguished visitors in the incubator for what would
become molecular biology. The Institut Pasteur was also an exciting
place where Jacob, Monod, Lwoff, Francois Gros, Jean-Pierre Changeux,
Giuseppe Attardi, and numerous bright young people worked in a ferment
of activity.
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NIH Again |
I returned to Bethesda in 1962 to become a section chief at
NIH in a group Gordon Tomkins had formed, the Laboratory of Molecular Biology. My research focused on the regulation of the histidine operon
(the cluster of genes involved with the biosynthesis of histidine in
Salmonella) and the role of transfer ribonucleic acid (tRNA)
in this regulation. I was very lucky in that the first three
postdoctoral fellows who came to my laboratory were Gerry Fink, John
Roth, and Robert G. Martin, a tremendously talented and enthusiastic
group. Among the significant contributions during this period was a
paper Bob Martin and I wrote on using sucrose gradient centrifugation
to determine the molecular weight of enzymes (7). Bob was a medical
student at Harvard who had come to my laboratory for a semester as part
of an NIH program for medical students interested in biomedical
research. I was enamored of the idea that the histidine biosynthetic
enzymes were in a complex in the cell, and I encouraged Bob to see if
this was true, using sucrose gradient centrifugation, a method that had
been developed for analyzing ribosomes and larger molecules. He worked
out a method for doing this during his semester at NIH. Though there was not much to the idea of a complex of histidine biosynthetic enzymes, our paper on the method became one of the most cited papers in
biochemistry. Bob enjoyed his time at NIH so much that he came back
after he finished medical school and stayed on with me as a
postdoctoral fellow. He later showed that the histidine biosynthetic
genes were turned on and off as a unit and that a single mRNA was
produced from the cluster of genes. His wife, Judith Martin, got a job
as a reporter at the Washington Post, and she eventually
became "Miss Manners," the columnist.
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The Test for Mutagens |
Sometime in 1964, I read the list of ingredients on a box of
potato chips and began to wonder whether preservatives and other chemicals could cause genetic damage to humans. I had been working on
some aspects of mutagenesis with Harvey Whitfield (8, 9). I thought it
would be useful to have a test for chemical mutagens and so I decided
to develop one. Because we had thousands of mutants of
Salmonella that required histidine for growth, mainly
isolated by Phil Hartman over the years, I made use of them in my
experiments. The experiments involve placing a few hundred million
bacteria onto a Petri dish containing agar medium with a trace amount
of histidine. This small amount of histidine allows all of the plated histidine-dependent bacteria to undergo a few cell
divisions. After the histidine is depleted from the medium only those
bacteria that have mutated back to wild type continue to grow and form visible colonies on a light lawn of the mutant. The spontaneous mutation rate for each strain is relatively constant. However, when a
mutagen is added to the assay mixture, there is an increase in the
number of histidine-independent colonies and a dose-response curve can
be obtained. During the next few years I developed a set of the most
sensitive tester strains using all of the known mutagens I could get my
hands on; I further improved the sensitivity of the test by eliminating
some DNA repair systems in the strains (10).
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Leaving NIH |
I was happy at NIH; Giovanna and I had two children, and we
liked the area. NIH was a great place to work, and we had a wide circle
of friends. What prompted my moving was that Gordon Tomkins, the
director of the Laboratory of Molecular Biology in which I worked,
started looking at job offers. Tomkins, who had both an M.D. and a
Ph.D., was an immensely bright polymath with extraordinary charisma,
charm, breadth, and intelligence. He was making a mark in science and
was known to everyone at NIH. Among the section chiefs in his
laboratory were David Davies, Marty Gellert, Gary Felsenfeld, Todd
Miles, and myself. Tomkins was at the center of our little universe,
but he was getting offers to become department chair at one university
or medical school after another and was seriously thinking about
moving. I became convinced that he was likely to accept one of these
offers and that our tight and compatible group would likely break up. I
mentioned to my friend, Jesse Rabinowitz at the University of
California, Berkeley that I might be on the market. Berkeley offered me
a job soon after, and as I always had a soft spot for California since
graduate school, Giovanna and I decided to move. I had been at NIH for
15 years. Tomkins moved to the University of California, San Francisco
soon afterward and we were close friends with Gordon and his wife
Millicent until his tragic early death. I felt privileged to have known
such an extraordinary fellow, as did almost everyone who knew him. I
have discussed his life in a commemorative essay (11).
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Berkeley |
I arrived at the University of California at Berkeley in
December 1967 as a Professor of Biochemistry. I continued to work on
regulation of the histidine operon with a series of graduate students
and postdoctoral fellows. I was particularly interested in the role of
histidine transfer RNA and its modified bases, which we had shown were
important in the regulation. In later years I also worked on the
regulatory system for defense against oxidants. I also continued my
work on mutagen detection, though for many years I considered it more
of a hobby until it became a major research focus. In trying to get
funding for the mutagen project I was turned down by the National
Cancer Institute (they did not think bacteria could teach us much about
cancer), but I finally got funded by the Atomic Energy Commission, as
they were interested in mutation.
My 30 some years at Berkeley were remarkably happy ones, and despite
some shiny job offers I never was tempted to leave. I enjoyed and
respected my colleagues, who were an amazingly responsible and
competent crew. I was fortunate in attracting excellent graduate students and a series of first rate postdoctoral fellows from all over
the world, aided no doubt by the allure of the San Francisco Bay Area.
The Biochemistry Department had a rotating chairmanship, and though I
tried to avoid the job for as long as I could, I served as the
department chairman from 1983 to 1989 out of a sense of duty. I am not
particularly good at administration (I am incorrigibly distractable and
find myself drifting off in committee meetings and thinking about
experiments) so I try to avoid administration whenever I can. I did,
however, form the National Institute of Environmental Health Science
Center at University of California, Berkeley in 1979 and served as the
director until 2002. There are now 22 such centers. I heard we were
known as the Center with poor administration but great science.
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Mutagens Again |
Because the mutagen project was viewed as more applied
research and was not basic enough for the graduate students and
postdoctoral fellows who came to my laboratory, I utilized mostly a
succession of wonderful undergraduate students who did honors work in
my laboratory. I have always had the policy of having about six or so
undergraduate students in the laboratory at any one time; I particularly enjoy their youthful enthusiasm. The word must have gotten
around that my laboratory was a good place to work so I think I
attracted some of the best undergraduates. Each undergraduate student
normally chooses a postdoctoral fellow to work with based on their
interest in the project. A high percentage of my papers from Berkeley
have undergraduate students as coauthors.
In the early 1970s we continued to improve the sensitivity of the
tester strains (12) and added to the mixture a liver homogenate fraction from rodents, which contains various metabolic enzymes (13).
Some chemicals are not mutagens themselves but become mutagens in the
presence of the liver homogenate, which can metabolize the chemicals to
an active form, which then mutates the bacteria.
I also became more and more interested in the relation of mutagens to
carcinogens. We showed that cigarette smoke was highly mutagenic (14)
and that we could detect most common chemical carcinogens with the
test, particularly after we added the liver homogenate (15). Having had
a background in genetics as well as biochemistry, all of my intuition
told me that mutagens ought to be carcinogens, though this was not the
prevailing view at the time or for many years. I became a proselytizer
for this view, though in retrospect I should have also emphasized the
role of cell division rates in mutation and carcinogenesis; I tried to make up for this later (16-19). It now seems obvious that increasing either DNA damage or cell division rates, e.g. by hormones,
increases cancer rates.
In contrast to the expensive and time-consuming rodent cancer test, our
method of assaying the mutagenicity of chemicals was simple, rapid, and
inexpensive. As a result, it was quickly adopted by thousands of
laboratories worldwide, particularly by drug and chemical companies,
for the detection of mutagens and potential carcinogens. Our method
made it possible to weed out mutagenic chemicals inexpensively early in
their development before they were introduced into commerce. I never
patented the test, in part because I thought it might detract from my
effectiveness in promoting mutagen testing, though I did have a brief
pang of regret when it seemed that almost every industry in the world
was asking for the strains. I started with the notion that industry
would be reluctant to use the test and that regulators would force them to. I soon realized that industry was eager to adopt the test, in part,
I concluded, because they had a huge incentive to weed out nasty
chemicals. Regulatory agencies only took notice of the test years
afterward, perhaps because a lack of competition created no incentive
to change their routine way of doing things. This experience, together
with subsequent interactions with bureaucracies such as the
Environmental Protection Agency and my readings in economics,
reinforced a growing conviction that to accomplish anything of
importance incentives matter.
Two major conclusions from our work were that mutation is one aspect of
the mechanism of cancer causation and that a high percentage of
carcinogens are detectable as mutagens. A series of wonderful students
did the work, especially Frank Lee and Bill Durston, particularly
brilliant undergraduates; David Levin, a graduate student; and Joyce
McCann, a postdoctoral fellow, who made a major contribution. An
extraordinarily competent and devoted laboratory technician, Edie
Yamasaki, also was a major contributor. I gradually drifted out of
bacterial work and into rats and humans. In recent years when people
ask me "Are you the Ames of the Ames test?" our work on the test
seems so long ago that I reply "Oh no, it was my father."
In the early 1990s we made one more improvement in mutagen testing. A
postdoctoral fellow, Pauline Gee, some students, and Dorothy Maron, a
laboratory technician, developed a new set of six strains that were at
least as sensitive as the old tester strains and also diagnosed the 6 possible base pair mutations (20). These strains not only showed
whether the test chemicals were mutagenic, they also indicated the type
of mutation. The University of California did take out a patent on this
improved test. In the beginning I thought that the mutagenicity test
would be outmoded in a few years, and I still find it surprising that people are using it 30 years later, despite all of the new genetic tools that have come along.
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Carcinogens |
My interests in cancer prevention and toxicology stemmed from
the mutagen test, and I soon became deeply immersed in both fields. I
enjoy learning new fields and I always seem to be in the midst of
learning a new one. I can often bring a fresh perspective to a new area
because of my broad interests in science. My laboratory at the time was
researching gene regulation and later regulation of antioxidant
defenses in Salmonella. I was helped enormously in entering
these new fields by finding a few extraordinarily intelligent and
competent associates who could help with the scholarship. A major find
was Lois Gold, who walked into my office after she saw a story in the
newspaper about a paper we had published in Science (21)
that Tris-BP was a mutagen. Tris-BP was one of the major flame
retardants, which a government agency had decreed were to be in all
children's pajamas. She was a mother with a young daughter and wanted
to know everything about flame retardants, burn statistics, risk,
evidence, etc. She was clearly unusually smart and thorough. After an
exhausting hour answering questions, I asked her what her background
was. She had a Ph.D. from Stanford and had a background in statistical
methods; she had taught at Berkeley and Stanford and was taking a few
years off to be with her child. She also had an interest in public
health policy. In my excitement about innovative ideas in new fields
there is a danger in getting carried away by enthusiasm, so I am always
looking for smart, tough minded associates who are willing to challenge my assumptions and data. I hired Lois on the spot to work whatever hours she could put in on various projects, including the Carcinogenic Potency Database I had started.
I had started the Carcinogenic Potency Database after I realized, in
trying to compare mutagenic potency with carcinogenic potency, that no
one had ever systematized the literature on the quantitative aspects of
animal cancer tests. We also found that carcinogens could vary by a
million-fold in potency. I applied for a grant to set up the database,
but it was turned down as I did not have any experience in animal
cancer tests, statistics, or pathology. This was all true, but we
thought it was important to do, wanted to do it, and knew we would do
it as well as it could be done if we consulted the best people in the
various fields, so we decided to go ahead anyway. Lois Gold stayed on
to develop the database, which is now the definitive quantitative
database in the world on animal cancer tests. Together Gold and I have written over 100 papers based on our analyses of the database. We have
challenged most of the assumptions in the field, so we have engendered
reams of controversy.
One important finding to come out of our analysis was that over half of
all the chemicals tested, whether natural or synthetic, were
carcinogenic when tested chronically in rodents at the maximum tolerated dose (MTD), the standard procedure in the rodent cancer bioassay (22). Our analysis suggested that carcinogenesis in the high
dose rodent tests was due to the use of a high dose and that the high
dose could cause chronic cell killing, inflammation, and cell
proliferation, which could account for the carcinogenic effects. We
concluded, therefore, that the tests did not provide information to
calculate low dose risks (18). These conclusions did not endear us to
scientists who have spent their lives testing synthetic chemicals at
the MTD, or environmental activists who have tried to purge the world
of tiny traces of synthetic chemicals, or regulators whose jobs depend
on eliminating traces of "toxic chemicals." I have become inured to
ad hominem attacks on Gold and myself that allege we are a
tool of industry, despite the fact Gold and I have always had a policy
not to accept money from industry, or to testify in lawsuits, or to
consult. It is clear that our critics do not like our conclusions, but
we have seen no convincing rebuttal of our science.
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Natural Chemicals, the Forgotten Control |
One observation that struck me fairly soon after our
mutagenicity test became widely used was the high rate at which we and others were finding mutagens in the natural world of plant chemicals. That got me thinking about the natural chemicals that humans ingest, such as the natural pesticides plants produce to kill off insects and
other predators (23) and the burnt material in cups of coffee (24).
Because almost all of the chemicals that humans are ingesting are
natural (23, 25) it seemed very improbable that synthetic chemicals
were likely to be more than a tiny fraction of our total exposure to
mutagens/carcinogens, other than from high dose occupational exposures
or medicinal drugs.
To put synthetic carcinogens in perspective, Gold and I thought it
necessary to examine the carcinogenicity of the natural background of
chemicals as an appropriate control for synthetic chemicals. We
estimated that 99.9% of all chemical exposure is from ingesting
natural chemicals in food, e.g. 99.99% of exposure to
pesticides is from ingesting natural pesticides produced by plants
(23). We published a paper entitled "Ranking Carcinogenic Hazards"
in Science in 1987 (17). To compare the average daily dose
of chemicals which humans might receive with the dose that induced
cancer in rodents, we created an index called HERP (human exposure
dose/rodent potency dose). The results of HERP showed that the possible
cancer hazard of traces of synthetic chemicals such as pesticide
residues are tiny compared with natural chemicals in the diet. Even the
possible hazards from "rodent carcinogens" in natural chemicals
should be viewed with skepticism because of the problem of high dose testing.
We also pointed out that diversion of resources and attention from
programs that focus on major risks to those that focus on minor
hypothetical risks might hurt public health. As I have become more and
more concerned with cancer prevention, I have concluded that we must
concentrate on major risks if we are to make any progress and that
concern with hundreds of minor, hypothetical risks is a distraction
from major risks, such as unbalanced diets and cigarette smoking.
Epidemiology is fraught with difficulties. "In Miami, study finds
everyone born Hispanic, dies Jewish." Epidemiology is useful when
there are large risks, but lacks the power to provide convincing
evidence that traces of synthetic chemicals cause small amounts of
human cancer. Though I am passionate about cancer prevention, I remain
skeptical of the purported dangers from traces of synthetic chemicals,
such as pesticides, and do not see much plausibility from either
toxicological or epidemiological analysis. Spending time debunking the
dubious assumptions (26) behind the environmentalist fervor against
traces of industrial chemicals does not prevent any cancer. Having
demonstrated the implausibility of such assumptions, I turned to
finding more effective ways to prevent cancer.
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Diet and Health |
In reading what was known about cancer prevention I was
attracted to the idea that unbalanced diets are a major contributor to
cancer because all the leading epidemiologists thought dietary factors
were likely to be in the same league as smoking. My intuition told me
there was a lot of interesting science in the diet-cancer area and that
the field was murky enough so there were not many people exploring it
(27). When I enter a new field, which I seem to do fairly often, I
always spend a fair amount of time getting an overview to see where the
least amount of effort will bring the maximum return, i.e.
exploring several different approaches with potentially high payoffs. I
am reluctant to enter, or stay in, a field that is very active, as I
find it too difficult to focus on just one thing. I think that my
talents lie in my finding new ways of looking at a problem and opening
up new fields, which is a result of my broad scientific reading and
interests. This, of course, makes it much harder to get grants. I was
fortunate to have received a large NCI Outstanding Investigator Grant
for 15 years, at which point NCI stopped that type of grant. The grant was made for me as it permitted me to do work on whatever interested me, and that saved my neck. I am enormously grateful someone had the
vision to fund such a program, at least for a while.
I was intrigued by the reviews of Potter and Block on 200 or so
epidemiological studies, which showed that the quarter of the
population eating the fewest fruits and vegetables had about double the
cancer rate for most types of cancer, compared with the quarter eating
the most. Our work on endogenous oxidants as a major source of DNA
damage had interested me in antioxidants, and I began to view vitamin
C, vitamin E, selenium, and other vitamins and minerals, many of which
came from the fruits and vegetables in the diet, as anti-mutagens and
anti-carcinogens (27). I began to think that much cancer in certain
human populations might be because of the less than optimal amounts of
anti-carcinogens and protective vitamins and minerals consumed in the diet.
The work that finally got me seriously involved in this area was the
research of Jim MacGregor on folic acid. Jim was a cytogeneticist who
spent a year in my laboratory (28). While assaying chromosome breaks in
humans and in mice, he stumbled on the fact that folic acid deficiency
breaks chromosomes in mice, just as radiation does. He then showed that
a person with a very high level of chromosome breaks was
folate-deficient and that a folate intervention lowered the level of
breaks. Folic acid comes from the Latin word folia (i.e. leaf); one gets it from green leafy vegetables. (My
graduate mentor, H. K. Mitchell, first discovered folic acid and
isolated it from 4 tons of spinach.)
Because low folate levels were very common in the population of the
United States, I talked a graduate student, Ben Blount, and then a
postdoctoral fellow, Matt Mack, into investigating the mechanism. They
showed that folate deficiency causes a block in the methylation of dUMP
to dTMP, which results in the misincorporation of millions of uracils
into the DNA of each rat cell, which causes chromosome breaks (29).
Removal of the uracil by uracil glycosylase causes a transient single
strand break (nick) in the DNA. Two opposing nicks, e.g.
from repair of a uracil across from an oxidative lesion, cause a double
strand break, the most serious DNA lesion. The chromosome breaks from
radiation are made by an analogous mechanism, the repair of opposing
oxidative lesions.
This prompted me to look into the whole array of vitamins and essential
minerals, as I think it likely that when one input in the metabolic
network is inadequate, repercussions will be felt on a large number of
systems and lead to degenerative disease. For example, deficiencies of
folate, B12, or B6 lead to an increase in DNA
damage and cancer (30); iron deficiency leads to neuron decay and
cognitive dysfunction (31) and mitochondrial decay and premature aging
(32). We have shown that inadequate levels of many vitamins or
minerals, such as iron, zinc, folate, B12, and
B6, result in DNA, mitochondrial, and other types of damage (30-35). Emily Ho, a postdoctoral fellow in my laboratory has shown, for example, that zinc deficiency in human cells in culture not only
fills the cell up with oxidants that damage DNA but disables p53, a
zinc enzyme, and also various other components of the DNA defense
network (33). Low intake of each of these vitamins and minerals is
found in 10% or more of the population, particularly in the poor
(30).
I am convinced that by tuning up metabolism by ensuring vitamin and
mineral adequacy we can effect a major improvement in public health
(35), particularly for the poor. Numerous efforts and programs to
convince people to change their diets have not been particularly
successful. Fortification, e.g. the folic acid fortification
of flour, has a role to play. Vitamins and minerals are amazingly
inexpensive; a multivitamin/mineral pill containing the recommended
daily allowance (RDA) for the essential vitamins and minerals costs
less than a penny to make. In fact, I think that everyone should take
one every day as "insurance" (35), though of course efforts to
encourage eating a balanced diet should continue. Vitamin and mineral
adequacy is important but is not the only part of our dietary needs,
which also include fiber, essential fatty acids, and other components
not found in a multivitamin/mineral pill (36).
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Oxidants |
I became interested in the early 1980s in oxidants from
metabolism, smoking, and chronic inflammation as a major source of mutagens; I started working in this area both in Salmonella
and in higher organisms (37-41). With some wonderful graduate
students, particularly Gigi Storz, Lou Tartaglia, and Mike Christman,
and postdoctoral fellows Robin Morgan and Fred Jacobson, we clarified for the first time the strategies employed by bacteria in their response to oxidants such as hydrogen peroxide (42, 43). The discovery
of the oxyR regulatory protein, which involved isolating it and
determining its sequence and DNA-binding site, provided general
insights into which cell constituents are damaged by oxidants and how
cells sense and respond to oxidative stress. It also showed that an
oxyR thiol operated directly as a sensor of oxidative stress
(44). A series of studies showed that oxyR controls a variety of genes,
including those that code for catalase and a new enzyme, alkyl
hydroperoxide reductase (42, 43, 45, 46), which was later cloned and
sequenced. Studies on the oxyR regulon led to the elucidation of the
mechanisms by which exposure of bacterial cells to low doses of
oxidants allows these cells to adapt to subsequent challenges of higher
doses of oxidants. These studies also provided insights in
understanding how higher organisms such as mammals adapt to oxidant
exposure. Work by Rhee and Stadtman identified the mammalian
counterpart to the alkyl hydroperoxide reductase. Other laboratories
have since identified similar oxidant-responsive elements in mammals.
We documented that endogenous oxidants from normal metabolism are
important in damaging DNA in both bacteria (47) and mammals (41, 48).
At one point, I had the vision that the lesions that were excised from
DNA by repair enzymes should be excreted in the urine and thus could be
analyzed. With the help of several sensational postdoctoral fellows,
including Mark Shigenaga, Robert Saul, and Rick Cathcart, we looked for
known oxidized DNA bases (the radiation biologists had worked out the
chemistry) in rat and human urine as a measure of oxidative DNA damage
(41, 48-50). This work suggests that there is a large rate of
endogenous oxidative damage to DNA (about 100,000 hits/cell/day in the
rat) (51). Moreover, though repair is very effective, some oxidative
lesions escape repair, the steady state level of oxidative lesions
increases with age, and an old rat has accumulated about 66,000 oxidative DNA lesions per cell (51).
Two extraordinarily good Swiss postdoctoral fellows, Roland Stocker and
Balz Frei, came to my laboratory to work on oxidation in the late
1980s. They and other students clarified the role of various
antioxidants in human plasma (52, 53) and discovered some major
antioxidants that were previously not fully appreciated, including uric
acid (37), bilirubin (54-56), and ubiquinol (57, 58). We showed that
ascorbate serves as a first line defense in blood plasma against lipid
oxidation (53) and as a key protective agent against oxidative damage
to sperm DNA (59). In the course of this work we developed many new
methods for measuring oxidative damage and defenses in tissues, as well
as in biological fluids such as urine and plasma. Throughout my career
I have always felt that developing new analytical methods helps to open
up a field and is well worth the effort. I take some satisfaction in
knowing that many of the methods we have developed are among the most highly cited papers. Because mitochondria are the main source of
endogenous oxidants and mitochondria are the main targets of oxidants,
this led directly to our work on the mitochondrial decay of aging.
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Delaying the Mitochondrial Decay of Aging |
Aging has been a major interest of mine for some time, in
particular the role of mitochondrial decay as a major contributor to
aging and age-related degenerative diseases. Mark Shigenaga and Tory
Hagen, two brilliant senior postdoctoral fellows, and I wrote a review
in 1994 (60) on why mitochondrial decay due to oxidant leakage from the
electron transport chain was likely to be a major factor in aging. This
idea was not original with us; Denham Harman and then Jaime Miquel had
discussed the free radical theory of aging, but we feel we contributed
some insights in the review. Writing the review got us all fired up to
work on the subject, and Hagen figured out an experimental approach in
rats that worked; we showed that there is a large amount of oxidative
damage to the mitochondria and mitochondrial decay during aging (61).
Kenny Beckman, another unusually creative postdoctoral fellow, and I
also reviewed the free radical theory of aging (62). These radicals can
cause oxidative damage, which in turn contributes to mitochondrial
decay and degenerative diseases such as cancer, aging, heart disease,
cataract, and brain dysfunction.
In a series of experiments by Hagen, and later after he left to go to
Oregon State, by another excellent postdoctoral fellow, Jiankang Liu,
we made progress in reversing some of this mitochondrial decay in old
rats by feeding them the normal mitochondrial metabolites, acetylcarnitine (ALCAR) and lipoic acid (LA), at high levels (63-67). The principle behind this effect appears to be that with age, increased
oxidative damage to mitochondrial protein causes a deformation of
structure of key enzymes, with a consequent lessening of affinity (Km) for the enzyme substrate (67). The effect of
age on the ALCAR binding affinity of carnitine acetyltransferase can be
mimicked by reacting it with malondialdehyde (a lipid peroxidation product that increases with age). Feeding the substrate ALCAR with LA,
a mitochondrial antioxidant, restores the velocity of the reaction,
Km for ALCAR-CoA transferase, and mitochondrial function. In old rats (versus young rats) mitochondrial
membrane potential, cardiolipin level, respiratory control ratio, and
cellular O2 uptake are lower; oxidants/O2,
neuron RNA oxidation, and mutagenic aldehydes from lipid peroxidation
are higher. Ambulatory activity and cognition decline with age. Feeding
old rats ALCAR plus LA for a few weeks improves mitochondrial function;
lowers oxidants, neuron RNA oxidation, and mutagenic aldehydes; and
increases rat ambulatory activity and cognition (as assayed with the
Skinner box and Morris water maze). I have been so excited about this work that I am sure it has added (or perhaps subtracted) a year or two
to my own life.
Two more outstanding postdoctoral fellows, Hani Atamna and Patrick
Walter, have shown that common micronutrient deficiencies accelerate
mitochondrial decay. Heme biosynthesis takes place predominantly in the
mitochondria. Interfering with heme synthesis causes a specific loss of
Complex IV with a consequent release of oxidants (68, 69). Iron
deficiency (25% of menstruating women in the United States ingest
<50% of the RDA) also causes release of oxidants and mitochondrial
decay (32) presumably through lack of heme (69). Vitamin B6
deficiency (10% of Americans ingest <50% of the RDA) also causes
heme deficiency (69). In a beautiful new paper Atamna shows that the
consequences are likely to be accelerated aging, neural decay, and
Alzheimer's disease (31).
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The Km in Genetic Disease, Polymorphisms, and
Aging |
One of my great pleasures in the last few years has been
working on an innovative, integrative review (70) with an undergraduate student, Ilan Elson-Schwab. We think this review will change thinking in both human genetics and nutrition. As many as one-third of mutations
in a gene result in the corresponding enzyme having an increased
Michaelis constant/Km (decreased binding affinity)
for a coenzyme, resulting in a lower rate of reaction. We review 50 human genetic diseases due to defective enzymes that can be remedied or
ameliorated by the administration of high doses of the B vitamin
component of the corresponding coenzyme, which we show raises levels of
the coenzyme and at least partially restores enzymatic activity
(70).
We also review five single-nucleotide polymorphisms in which the
variant amino acid reduces coenzyme binding and thus enzymatic activity; the reduced levels of activity are likely to be remediable by
raising cellular concentrations of the cofactor through high dose
vitamin therapy (70). Some examples of polymorphisms include the
(C677T; Ala-222
Val) methylenetetrahydrofolate reductase (NADPH)
and the cofactor FAD (in relation to cardiovascular disease, migraines,
and rages), the (C609T; Pro-187
Ser) mutation in NAD(P):quinone
oxidoreductase 1 (NQO1) and FAD (in relation to cancer), the (C131G;
Ala-44
Gly) mutation in glucose-6-phosphate 1-dehydrogenase and
NADP (in relation to favism and hemolytic anemia), and the
(Glu-487
Lys) mutation (present in about half of Asians) in
aldehyde dehydrogenase and NAD (in relation to alcohol intolerance,
Alzheimer's disease, and cancer). As all of the polymorphisms are
sorted out in humans, this Km concept may be
relevant for tuning up the metabolism of much of the population. I
suspect this might be one of the first major contributions of genomics to public health. We also are actively working on whether high doses of
some of the B vitamins might help delay the mitochondrial decay of
aging. To encourage further discussion and new information on this
topic we have set up a web site (www.KmMutants.org).
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Children's Hospital of Oakland Research Institute (CHORI) |
At the end of 1999 our building on campus, Barker
Hall, reached the top of the list of buildings that needed to be
renovated for earthquake reinforcements. We were asked to vacate the
building. I was told to cut my laboratory in half and that the
University would figure out where to squeeze me in for 2-3 years until
the renovations were completed. Instead, I said goodbye to campus and
moved to CHORI, with the encouragement of its director Bert Lubin.
CHORI is a nearby research facility that has recently renovated a
beautiful old high school and turned it into laboratories. We recently
occupied a newly renovated wing, which will be a Nutrition-Genomics Center within CHORI. I have been exceptionally happy here these last
few years in the company of first rate colleagues, and I do not plan to
ever move again.
I recently told a colleague that I thought I was doing the best work of
my scientific career. He replied, "Bruce, you've been telling me
that for 30 years." Thus, aging has not damaged my enthusiasm genes,
though I am not as certain about my neurons. My current passion, as can
be seen from the above sections, is tuning up metabolism in humans,
both in the young and the old, by vitamins, minerals, and biochemicals.
I think this will lead to a marked improvement in health and an
increase in longevity. With so much work to do, I have no plans to
retire from science.