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Vol. 03 Issue 3, Summer 1998

Contributions of Experimental Animal Research to the Understanding of Human Breast Cancer
The Ribbon 

What causes breast cancer? Is there something that can prevent it, or cure it? Researchers are using many different approaches and sophisticated techniques to answer these questions. The current fast-paced and highly technical world of scientific research makes it very difficult to keep up with all of the new information. Some knowledge about the scientific process, experimental design, and data interpretation may provide non-scientists with an appreciation for and greater understanding of breast cancer research.

There are many different ways to study breast cancer. One way is to examine the association between the disease and various biological and environmental factors through epidemiologic studies. The key features and specific examples of epidemiologic studies were recently reviewed in Volume 3, Number 1 of The Ribbon. Other types of studies rely on animal models and/or isolated breast cells or other kinds of cells grown in the laboratory (cell culture studies). Laboratory studies that use animal models of breast cancer are the focus of this article. Look for an article on cell culture and similar studies in a future issue of The Ribbon.

All basic scientific studies follow a similar plan. They start with a hypothesis, based on previous observations and the results of other animal, cell culture or human studies. A hypothesis is an assumption that a scientist makes. The next step is to design an experiment to test the hypothesis. There is no general set of guidelines to follow for designing a good scientific experiment, and what is considered "good" depends on the type of experiment being done. In this article, the important features of two different kinds of animal experiments are discussed, as well as how they contribute to the understanding of human breast cancer.

Animal studies are an important part of breast cancer research because they offer scientists a way to ask questions about cause, prevention and treatment in a controlled environment. In epidemiologic studies, scientists have to account for natural differences in their sample of the human population. For example, there are individual genetic differences among people that may account for how an individual reacts or responds to a carcinogen or to a cancer treatment. Also, the scientists may not know what people were exposed to during periods of their lives outside of the experimental time frame. Finally, it is not possible or ethical to control all aspects of a person's diet and/or lifestyle choices or to expose her/him to a suspected carcinogen. In animal studies, scientists have more control over all of these situations and this is a major advantage.

Although there are some obvious biochemical and physiologic differences between animals and humans, there are more similarities. Therefore, although results from animal tests are never directly extrapolated to humans, animal and human cancer studies are closely related to and dependent on each other. Advances in the knowledge of breast cancer are made by combining the results from animal and human, as well as cell culture, studies.

Scientists use animal models of breast cancer in many different ways including to: 1) test the carcinogenicity of a chemical; 2) determine if a chemical acts like the hormone estrogen; 3) determine if a particular food or a natural chemical in that food may prevent the development of breast cancer; 4) test if a new drug is an effective treatment for breast cancer; and 5) learn more about the basic molecular and biochemical mechanisms underlying the cancer process. An animal study designed to test the effectiveness of a new anti-cancer drug is discussed in the "Research Commentary" section of this issue. In this article, two other types of animal studies, the long-term cancer bioassay and nutrition studies, are discussed. For the cancer bioassay, we highlight some of the critical features required to make this type of experiment valid. For the nutrition study, we use an example to illustrate one approach to this type of experiment.

The Cancer Bioassay

Scientists use the cancer bioassay to determine if a particular chemical is a potential carcinogen. Because of the importance of this type of study to human health, many different organizations such as the World Health Organization (WHO), the Environmental Protection Agency (EPA), the International Agency for Research and Cancer (IARC), and the National Toxicology Program (NTP) have contributed to the development of guidelines for the design and interpretation of experiments to test the carcinogenicity of a chemical. A study designed to test the carcinogenicity of a chemical must follow these guidelines in order to be considered an appropriate cancer bioassay.

In the U.S., the majority of cancer bioassays to test for a pesticide's cancer causing potential are either conducted by the NTP, or through industry contracts established to meet the EPA¹s requirements for health effects assessment for pesticide registration. Most of these studies are not published in the scientific literature. However, abstracts of the NTP studies are available on the web: http://ntp-server.niehs.nih.gov/main_pages/NTP_ALL_STDY_PG.html. Organizations such as IARC and the EPA analyze the results of all relevant studies before evaluating a chemical¹s carcinogenicity. BCERF offers a unique assessment of chemical carcinogenicity with specific reference to breast cancer through critical evaluations of the research on pesticides and other chemicals. Some of the requirements of the cancer bioassay that BCERF considers when evaluating a study are described below.

Cancer bioassays must be conducted in accredited laboratories to ensure that proper animal care, housing, and feeding are taking place. A cancer bioassay should be conducted in more than one animal species ‹ typically mice and rats. In each of these species, both male and female animals should be included. Finally, the cancer bioassays require a larger number of animals compared to other animal experiments. Usually at least 50 animals per dose are needed to do the proper statistical analysis for tumor incidence.

There are other important considerations regarding the design of the cancer bioassay. First, the scientists must correctly administer the chemical to the animals using the route most likely experienced by a human population. Two acceptable routes of administration include ingestion and inhalation. Sometimes it is acceptable to apply the carcinogen to the skin. Injection of the animal with a carcinogen is not an appropriate route of administration and may cause other side effects.

Different doses of the potential carcinogen are used to see if there is a dose-response effect and to generate a dose-response curve for cancer risk assessment. One group of animals does not receive any of the carcinogen and they serve as the controls. It is particularly important that the control animals are obtained from the same facility as the experimental animals and that they are the same strain and age. In addition to the control group, there are at least three treatment doses. The highest dose needs to be the "maximum tolerated dose" (MTD). This dose is defined as the dose required to cause a 10% to 15% decrease in body weight gain without significantly affecting the survival of the animals.

The animals in the cancer bioassay need to be exposed to the potential carcinogen for at least 24 months to allow enough time for tumors to develop. After tumor formation, it is important for the scientists to evaluate them using pathologic criteria; for example, to determine the incidence and type of benign and malignant tumors.

Definitions

In vivo: in the living body. An experiment in an animal model is referred to as an in vivo experiment.

In vitro: in an artificial environment. An experiment in a test tube or cell culture system is an in vitro experiment.

Dose: refers to the concentration or amount of a drug, chemical or food given to an animal (in vivo) or added to cells in culture (in vitro).

Inbred strain of rats: a population of laboratory rats derived from a small set of ancestors. These animals are more closely related genetically than if mating had occurred by random selection.

The cancer bioassay is an important method to identify potential human carcinogens. Regulatory agencies use the information obtained from animal cancer bioassays in combination with results from other experiments, such as epidemiologic studies, to evaluate the cancer causing potential of a chemical or pesticide.

The Organization of a Scientific Paper After completing a study, a scientist usually communicates the results to the rest of the scientific community by writing a research paper. These papers are reviewed by other scientists before publication. Most of these papers are organized as follows:
Introduction: In this section, the scientist provides background information to place his/her work in the proper context. Also, the hypothesis being tested is usually stated here.
Methods: This section contains a description of the experimental design. It usually contains details of all the materials and procedures used.
Results: The scientist will describe the results of the experiment using words, graphs and pictures when necessary.
Discussion: In this section, the scientist will attempt to explain the results of the experiment. This may involve discussing any limitations of the study as well as putting the results into the context of other previous studies described in the introduction. Then some conclusions can be proposed and suggestions made for future studies.
References: The scientist will list the previous studies that he/she referred to throughout the paper.

Nutrition Studies

Information from several different types of epidemiologic studies has indicated that diet may be very important for both the prevention and treatment of breast cancer. This observation has led to hypotheses and experiments using animal models of breast cancer to specifically test various components of the human diet.

Example: Brussels sprouts

The investigators in this study (Stoewsand et al., 1988) hypothesized that brussels sprouts are protective against mammary carcinogenesis (breast cancer). In contrast to the cancer bioassay, there are no established guidelines to follow for the experimental design of an animal nutrition study. Although these types of studies are important in the field of breast cancer and other chronic diseases, they are not contributing information to help determine whether or not a chemical is a health threat to human beings. Therefore, scientists who conduct these studies are not held to the same set of rules and are more free to creatively investigate methods of prevention.

To test their hypothesis, the scientists used 60 female Sprague-Dawley rats. This is because mammary tumors in rats mimic breast cancer in humans and the tumors are similarly affected by genetic, hormonal, dietary and environmental factors. Scientists use inbred strains of rats, such as Sprague-Dawley, because they are very genetically similar.

In this type of study, there is no specific number of animals required as in the cancer bioassay. Instead, scientists choose a number of rats based on their calculation of the number necessary to generate sufficient statistical power. This is also important in epidemiologic studies. As in the carcinogenic bioassay, all of the rats were the same age, and had the same access to food and water. The scientists chose to induce the formation of mammary tumors by using a drug called 7,12-dimethylbenz[a]anthracene (DMBA), a synthetic chemical used in experimental studies that is known to cause mammary tumors. By using this method, the scientists did not have to wait for tumors to develop spontaneously or naturally, and they knew when the mammary tumors were initiated.

The rats were divided into 4 groups of 15 rats each as follows: 1) Group A was fed a diet that consisted of 20% dried brussels sprouts, and they received a dose of DMBA; 2) Group B was fed a diet that consisted of 20% brussels sprouts, and they did not receive any DMBA; 3) Group C was fed a regular rodent diet, and they received a dose of DMBA; and 4) Group D was fed a regular rodent diet, and did not receive any DMBA. Several weeks into the experiment the diets were switched, and Groups A and B were fed a regular diet while Groups C and D were fed a diet that consisted of 20% brussels sprouts. This experiment was designed to determine if and when during the cancer process ‹ at initiation, promotion, or progression ‹ brussels sprouts would be most effective. The rats were weighed during the experimental time frame because it is important to determine if the dietary intervention also affected their growth and weight. The scientists checked for tumor formation by touch, but then confirmed their reports by looking at the tumor cells under a microscope and defining them by pathological criteria.

The researchers found that the rats who were fed brussels sprouts early in the experiment were less likely to develop cancerous tumors as compared to the rats fed brussels sprouts only late in the experiment. There was some evidence that the mammary tumors shrank in the rats fed brussels sprouts later in the experiment. This suggests that brussels sprouts are very effective early in the cancer process when the tumors are being initiated. They are somewhat less effective in preventing tumor progression or promoting tumor regression. The investigators also reported that there was an increase in the number of benign tumors in the rats fed brussels sprouts early in the experiment as compared to later in the experiment.

This study provides some good and positive information on the cancer preventing potential of brussels sprouts, but also leaves some questions unanswered. To put the results in the proper context, there are some limitations of the study that need to be considered. For example, rats don¹t normally eat brussels sprouts and the diet of the animals fed the brussels sprouts may be different in several ways from the diet of the animals fed the regular rodent chow. When the scientists added the brussels sprouts, they had to remove some other dietary components. Also, the scientists only looked at one concentration or dose of brussels sprouts, 20%. Future experiments might try to determine if a different dose of brussels sprouts would still be effective against the development of cancerous tumors, but not promote the formation of benign tumors.

The evidence from this study, future animal studies, and epidemiologic studies should be considered together before reaching any conclusions regarding the amount of brussels sprouts in a human diet needed to help prevent the development of breast cancer.

The results of both cancer bioassays and nutrition studies need to be considered within the framework of the strengths and limitations of the experimental method. Then this information should be combined with the results from human and cellular and molecular studies to design better prevention, treatment and diagnosis strategies for breast cancer.

Written by Julie Napieralski, BCERF Research Associate

Reference

Stoewsand, G. S., J. L. Anderson, and L. Munson (1988) Protective effect of dietary brussels sprouts against mammary carcinogenesis in Sprague-Dawley rats. Cancer Letters, 39: 199-207.

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Last Update 06.23.00