The Role of Cooked Food in Genetic Changes

CCORDING to a common, rather simplistic notion, we are what we eat. On a far more empirical level, epidemiological studies reveal a connection between diet and adverse health consequences. Many observed differences in cancer rates worldwide, including incidences of colon and breast cancer, are linked to variations in human diets.
Strong evidence suggests that mutations are the initiating events in the cancer process. In other words, the complex sequence of cellular changes ultimately leading to malignant tumors is thought to begin with structural changes--mutations--within the molecular units that make up the genes. For 17 years, LLNL researchers have been investigating certain biologically active compounds in foods that can trigger tumors in animals, at least after exposure to high concentrations, by producing cellular mutations.
At first glance, identifying the mutagens that might put us at risk and understanding how they affect the body appear to be simple matters. In fact, the opposite is true. Consider just a few of the questions that must be addressed to understand the entire picture of diet-induced mutations and possible links to cancer. Exactly what compounds in foods are dangerous, how are the compounds formed during cooking, in what amounts are they present after cooking, and how toxic or cancer-causing are they? What chemical changes take place metabolically at the molecular level after the mutagenic substances are consumed? For example, what role do metabolic enzymes play, how is DNA affected, and how might tumors be triggered in the body's somatic cells? What chemical, tissue, animal, and human models might be useful to estimate risk to the human population? Are all people affected similarly, or are some resistant to cancer-causing effects? If people vary in cancer incidence, what accounts for the differences in susceptibility?
Clearly then, isolating, identifying, and assessing the biological activity of mutagenic compounds in food is a difficult problem requiring extensive effort. Table 1 is an overview of some of the research issues addressed and analytic methods used in this field of investigation. This series of articles focuses on the first five questions under "Issues" listed in Table 1. A second installment in Science and Technology Review will address the remaining issues.
A simple analogy can help put a key feature of our work into perspective. The compounds we have been investigating for nearly two decades--the aromatic heterocyclic amines--are present in cooked foods at very low levels, in the range of about 0.1 to 50 parts per billion. Isolating material at the part-per-billion level is equivalent to pouring a jigger of Scotch into the hold of a full supertanker and then trying
to retrieve it again. Although the compounds we study are present in very small amounts, they are also the most mutagenic compounds ever found, and they produce tumors in mice, rats, and monkeys. Such knowledge, combined with the fact that these compounds are present in many foods characteristic of the Western diet and that certain diets are known to influence tumors at several body sites, gives our research an extra sense of urgency.

LLNL's Approach
The single aspect that best characterizes our research on food mutagens and carcinogens--and sets our work apart from almost all other efforts around the world--is its multidisciplinary nature. Biomedical scientists at LLNL routinely collaborate with investigators working in analytical chemistry, synthetic chemistry, quantum chemistry, physics, the environmental sciences, and forensics (Figure 1). Our research requires tools such as accelerator mass spectrometry and nuclear magnetic resonance spectrometry, to name a few. The Laboratory is one of the few places that brings together the broad expertise and state-of-the-art analytic tools required to fully understand each important aspect of the problem of mutagens and carcinogens in the human diet. The way we became involved in this field of research has much to do with our role as a national laboratory with interdisciplinary research programs.

Figure 1. Cyndy Salmon, one of the researchers in the LLNL food mutagen research group, pours a cooked food sample into an extraction tube to prepare it for subsequent analysis.

Mutagens are the damaging agents that can structurally change the molecular units that make up the genes (that is, the genetic material, DNA) or the relation of one chromosome to another. For many years, LLNL investigators have been studying some of the ways that x rays, ultraviolet light, and some chemicals in the environment can act as mutagens. Carcinogens are agents that incite the development of a cancerous tumor or other malignancy. Some 80 to 90% of mutagenic substances are also carcinogenic. More than 50 years ago, scientists painted the skin of mice with extracts from heated animal muscle and found that the extracts were carcinogenic, but the research went no further.
By the early 1970s, Bruce Ames at the University of California, Berkeley, had developed a biological test to measure the mutagenic potency (mutagenicity) of substances.(Reference 1) In the late 1970s, T. Sugimura, who directed research at the National Cancer Center in Tokyo, applied the Ames method and published the fact that smoke condensate from cooking and the charred surface of broiled fish and beef were mutagenic.((Reference 3)
The news that cooking amino acids (the building blocks of proteins) and muscle-containing foods could be dangerous triggered considerable scientific interest around the world. In 1978, biomedical researchers at LLNL were working on the problem of mutagenic chemicals produced by oil shale retorting and coal gasification. Because of our combined expertise in chemical analysis (including different types of chromatography and spectrometry), biological analysis (including the Ames mutation assay), and our emerging program in genetics and toxicology, we received a multiyear contract from the National Institute of Environmental Health Science (NIEHS) to take a detailed look into the problem of food mutagens. As it turns out, what happens when oil shale and coal are heated is not so different from some of the chemical processes that occur when a hamburger is cooked.
Our work on food mutagens also parallels our interest in the mechanisms by which pesticides and many other toxic chemicals can elicit adverse biological responses. For example, benzo[a]pyrene is a widely studied pollutant found in combustion products, and it has been isolated from burned fat and cigarette smoke. However, this compound becomes carcinogenic only after it interacts with DNA following oxidation by metabolic enzymes. The production of such enzymes and their roles in changing the chemical reactivity of compounds are part of the body's normal biological response to certain foreign substances. We are learning that similar "metabolic activation" takes place before food mutagens become harmful.
Today, our research is funded primarily by the National Cancer Institute, with additional support from the Laboratory Directed Research and Development program and other sources. There are approximately 50 other prominent research teams worldwide studying the heterocyclic amines. However, except for one other program in Japan, ours is the only team that brings a truly multidisciplinary approach to the problem of understanding mutagens and carcinogens associated with cooked food and their consequences at the cellular, genetic, and molecular levels.

A Problem of Strategy
Strictly speaking, it is inaccurate to say that cooked foods contain mutagens. More precisely, certain cooked foods contain premutagenic substances (promutagens) that are metabolized by enzymes naturally present in body tissues, leading to the formation of one or more reactive mutagenic substances. Conventionally, however, "promutagen" and "mutagen" are used synonomously, and we have followed that practice here unless the point being made about the research demands a precise distinction.
At the outset of our research, we were faced with problems of strategy. Studying substances that are present at very low concentrations imposes many research constraints. If we focused on only a few foods, as seemed wise, then our results and their implications for public health might be misinterpreted. Instead, we decided to examine the foods that are the principal sources of cooked protein: meats (any muscle-containing food, including fish), eggs, beans, cheese, and tofu. Whereas we initially focused on meats, especially fried beef, we have now expanded the range of foods to include cooked breads and grain products, heated flour from many different plant sources, and meat substitutes.
Over the years, our research has also evolved from relatively simple concepts and approaches to more sophisticated ones. Initially, we had to identify the mutagenic compounds in heated foods because many were not known (that is, neither synthesized nor analyzed). Thus, we focused our efforts on identifying the chemical composition and structure of mutagens, assessing how different cooking procedures affect the formation of mutagens, and determining the amount (abundance) of the mutagenic products. Even though chemical identification and quantification are still important activities, our work has expanded to include many other aspects of the problem.
For example, we developed techniques to help us learn how mutagens are metabolized in the body. We use animals as models to understand complex metabolic pathways and are developing cell-culture methods that model human metabolic systems. One particularly important issue is how metabolites (the intermediate products formed by enzymes) interact with the genetic material. We need to know exactly what takes place at the molecular level, including covalent binding with and structural changes to specific components of DNA. This work taps the skills and facilities in several related research programs, including the Human Genome and DNA repair projects. (See the April/May 1992 and April 1993 issues of Energy and Technology Review for more background on these two programs.)
In assessing the effects of low-level exposure to food mutagens, we make use of Laboratory expertise in accelerator mass spectrometry (AMS). Yet another part of the story is the differences among humans in susceptibility to cancer, which has become our newest effort. In essence, our success in recent years is derived not so much from simply applying standard analytical methods by themselves as from combining biological analysis with state-of-the-art analytical tools available at LLNL to study all aspects of the health risks, ranging from dietary exposure to effects in model systems and humans.

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