Experimental Studies in Animals (In Vivo)
Definition: The use of non-human animals in scientific experiments
Experiments involving rats and mice have deepened scientific understanding of complex changes in breast tissue over the lifespan, which is essential for examining the effects of chemical and radiation exposures at critical periods of mammary tissue development. A convening of 75 mammary gland biologists, toxicologists, pathologists, epidemiologists, risk assessors and regulators concluded in consensus that, “Given what we know about human, rat, and mouse mammary tissue development progression, the rat and mouse are adequate surrogates for human breast development” (Rudel, 2009b).
Atrazine enhances the activity of the enzyme aromatase, which converts testosterone to estrogen in bodily tissues. Ecological studies of atrazine have found evidence of endocrine disruption of animals in their natural habitat. In particular, Hayes (2002) found dramatic changes to reproductive development in frogs exposed to low doses of atrazine, including abnormal internal and external reproductive structures, and abnormal adult hormone profiles Laboratory studies in rats (Enoch, 2007; Raynor 2005) have found that low-dose exposures to atrazine or atrazine metabolites during fetal development lead to delayed mammary gland development.
A study that exposed rats to BPA from early pregnancy through weaning found that the offspring had mammary gland adenocarcinomas at 90 days of age, indicating that BPA might act as a complete carcinogen – not needing any other factor to cause tumors (Acevedo, 2013). This unexpected result was not predicted from previous in vitro or genomic experiments and illustrates how animal experiments can provide evidence that is currently not available using other methodologies.
The estrogen system and mammary gland development of rodents parallel those of humans, making mice and rats good models for studying effects of early exposures to environmental toxicants on susceptibility to tumor development. As noted in the 2013 Report of the Interagency Breast Cancer and Environmental Research Coordinating Committee, “The integration of animal and human research offers the best opportunity to understand the contribution of environmental factors to breast cancer risk, the underlying mechanisms, and the potential for prevention strategies.” (Ch 5., pg. 2)
Use of animal models allows scientists to control the level, timing and combinations of exposures to environmental chemicals at identified periods in the animal’s development. Such intentional dosing of humans runs counter to established scientific ethics. Animal studies have demonstrated changes in gene expression associated with development of breast cancer (Chan, 2005; Drost, 2009; Shoushtari, 2007) as well as some of the interactions between genetic and environmental factors in altering risk for breast cancer (Zarbl, 2007).
Currently, animal studies retain an advantage over purely cell-based in vitro studies, as growing evidence indicates that cancer cannot be explained solely by an accumulation of genetic mutations cells or tissues. Changes in the development of and interactions between different cell types may predispose an animal (including a human) to cancer (Maffini 2005; Sonnenschein, 2000). This shift in focus toward examining the more complex biological context in which cancer develops is essential to our understanding of the ways that environmental factors affect molecular, sub-cellular and tissue organizational systems, and lead to greater breast cancer susceptibility.
Rodents’ short lifespans present a limitation given the long latency between exposures and diagnosis of breast cancer often observed in humans. Also, rates and processes of progression of mammary/breast tumors in rodents is significantly different than those of humans (Kim, 2004b).
In recent years, there has been considerable debate over whether or not injection of chemicals (such as BPA) into rodents mirrors human exposures, which tend to be mainly through diet, inhalation or absorption through the skin. In the case of BPA, a considerable literature evaluates the metabolism of the compound. These studies have examined differences in metabolism between species and tissues in response to varying doses administered in different ways (for example, through injection, consumption in food or water, or directly into the stomach or via the skin). The data suggests that all of these factors influence the pattern of internal body exposure to active forms of the chemical.
Species and strain differences
Not all rodents are equally sensitive to the hormone-disrupting effects of endocrine-disrupting compounds. Many strains of rodents used in biomedical research are inbred strains, and therefore have low genetic variability. Scientists can take advantage of the reliable and known differences between strains to test hypotheses about the relationship of particular environmental exposures and later development of mammary (breast) tumors. For example, strains of rats with low estrogen-receptor levels are relatively non-responsive to endocrine disruptors like BPA, in terms of later effects on reproductive and developmental processes, including effects on mammary tumor development (Ryan, 2010; Gray, 2010; vom Saal, 2010). As a result, to study effects of chemicals that might mimic estrogen, scientists can use estrogen-sensitive strains along with appropriate positive-exposure controls (like estradiol or DES exposure), to study the effects of exposure to BPA (vom Saal 2010). Ultimately, understanding the variability in sensitivities to exposures across species, strains and individuals will be critical in understanding the nuanced mechanisms by which environmental exposures affect risk for people with differing vulnerabilities.
Reducing the use of animals
The debate about the ethics of animal studies extends into the scientific community, where efforts are underway to reduce reliance on animals where possible. Where they exist, alternative testing methods that do not involve the use of animals and provide equivalent or superior scientific information should be used. Where high quality, validated non-animal tests do not exist, methods that use fewer animals than conventional studies but retain statistical power should be used, including the use of tests that combine multiple health endpoints. Results from such studies should be shared widely in order to avoid unnecessary duplication of research. Sustained investment is needed to develop new, high quality, validated non-animal tests so that the use of animals is reduced and eliminated in a reasonable timeframe.
Mammary gland changes Very few widely used chemicals have been studied for their effects on mammary (breast) tissue development or breast tumor development in exposed animals (Rudel, 2011). Traditional toxicology studies, which require that certain reproductive endpoints be examined to evaluate carcinogenicity, do not currently require examination of mammary tissue development. Yet mammary tissue is uniquely vulnerable to many of the chemicals to which we are exposed. Thus, studies that rely on less sensitive endpoints and do not find evidence of cancer causation may lead to false security that exposures are without consequence. Validation of accepted measurements that clarify what is normal and what is pathological development of mammary tissue (including tumor development) will be essential both for advancing scientific progress in this field, but also for informing regulatory decisions.