The Search For Alternatives To Animal Testing Intensifies

One need only read the labels on skin-care products--"not animal tested" and "cruelty free"--to realize that the animal rights movement has hit consumers in the heart, and manufacturers are responding to their concerns. While the rights and wrongs of animal research are still being fiercely debated, few scientists would deny the importance of finding substitutes for animals wherever possible. Not only is it desirable to minimize suffering of animals, but also massive amounts of paperwork and the costs of housing animals can be reduced if alternative technologies are found. A number of alternatives are already on the market, and still more are being developed. These include such products as artificial skin to measure irritancy and toxicity of various substances; a silicon microphysiometer, which measures individual cells' responses to physical and chemical stimuli; and even cultured organ systems, being developed to examine systemic reactions to substances. Two widely used tests illustrate that replacements for laboratory animals, or at least ways to minimize harm to them, were being sought before the animal rights movement began to receive media attention. A generation ago, a rabbit died for every human pregnancy officially diagnosed. Then, in the 1970s, monoclonal antibodies against human chorionic gonadotropin, the so-called pregnancy hormone, were developed. This approach required nurturing hybridoma cells in mouse abdominal fluid, resulting in the sacrifice of a much smaller number of animals as compared to the rabbit method. Monoclonal antibodies can now be mass-produced in genetically engineered bacteria. The Limulus amoebocyte lysate (LAL) test consists of freeze-dried horseshoe crab blood cell extracts that, when reconstituted with water, gel in the presence of bacterial endotoxin. The test is used to detect contaminated medical equipment and drugs, and replaces injecting suspect material into a rabbit to see if it causes a fever or death. But the crabs need not be sacrificed to provide the LAL test. Each summer, local Cape Cod fishermen round up the creatures, and truck them to Associates in Cape Cod Inc. in Falmouth. There, college students hang the crabs upside down and a technician pierces a muscle in the central hinge. About a third of each crab's metallic-blue blood is collected in large jugs that look like office water coolers, and the amoebocytes are separated out. The creatures are then trucked back to their beaches, apparently none the worse for their experience. In Vitro Tests A 1986 Office of Technology Assessment report delineated the goals of animal test alternatives, summarized in three "Rs": replacement, reduction (in numbers of animals), and refinement (lessening pain on the part of the animals). A test satisfying at least one "R" must then tackle "V"--validation. For every new procedure, known toxins are tested to see if they are ranked (by severity of effect) in the same order as in vivo protocols, such as the now-banned Draize rabbit eye irritancy test, would have ranked them. In vitro tests are designed to target the same questions asked when whole animals are used: Does the substance cause mutations or cancer? Skin or eye irritancy? Long-term chronic effects? Neurotoxicity? Is reproduction or development compromised? But, because of their specificity, many animal alternatives can zero in on cytotoxicity, even to the level of individual organelle responses, in a way not possible by subjectively judging the condition of a rabbit's eye or skin. In vitro tests can replace animals or reduce their numbers at various points: initial drug screening, analysis of receptor activity, design of analogs that tame toxicity, and postmarketing surveillance to monitor side effects. Skin-Deep Systems Several companies are pursuing skin replacement, both for in vitro toxicology testing and, farther down the line, as graft material for burn patients. The human cells in the Skin2 Dermal Model, from the La Jolla, Calif.-based Marrow-Tech Inc., come from the dermis of a newborn's foreskin tissue discarded after circumcision, seeded onto inert medical-grade nylon mesh. Here, the cells divide and metabolize, secreting a collagen matrix and growth factors, as they normally would. The Skin2 "Full-Thickness" model caps the dermis with an epidermis. The company's next product will be a stratum corneum (skin surface) model, which mimics the lipid barrier function of skin and is expected to be useful for evaluating penetration of skin-care products. Organogenesis Inc. of Cambridge, Mass., uses a variation on the foreskin theme for its Testskin by seeding human foreskin tissue into a bovine collagen gel. Nutrients are added, the cells secrete collagen and growth factors, and an epidermal layer is placed on top. Manufacturers now using Testskin include Est‚e Lauder Inc., Mary Kay Cosmetics Inc., Avon Products Inc., and Amway Corp. Next from Organogenesis will be a skin model containing pigment-producing melanocytes useful for testing sunscreens and makeup for dark-complexioned people, says John Burger, director of investor relations for the company. Organogenesis also offers a more versatile "living tissue substrate." Says Burger, "It can be custom-seeded with epithelial cells of whatever organ you want to mimic. For example, you can study the effects of a compound on the stomach lining by taking a sample of gut epithelium and seeding it on top of the substrate." But a substitute skin need not be multilayered to yield information. Clonetics Corp., based in San Diego, makes the EpiPack, a test kit for cell viability that uses a simple monolayer of human keratinocytes, grown without serum. EpiPack also includes supplies to conduct the neutral red bioassay, a very accurate and elegant test of cell viability developed by Ellen Borenfreund and colleagues in 1985 at the Laboratory Animal Research Center at Rockefeller University in New York. It is based on the fact that lysosomes (organelles containing digestive enzymes) of live cells absorb the vital dye neutral red, whereas dead cells do not. A substance's cytotoxicity, then, can be assessed by the intensity of the dye on a plate of cells or substitute skin. Marrow-Tech and other companies also offer neutral red bioassay kits to use with their substitute skin products. In a typical neutral red bioassay, cells are exposed to several dilutions of potentially toxic agents for 48 hours, then incubated with neutral red for three hours. The dye is extracted with a solvent and quantified in a microplate reader. The "endpoint" is the concentration of the agent that causes a 50 percent reduction in dye uptake. The endpoints for several known toxins appear in the same order in the neutral red assay as they do on the Draize test--that all-important validation. Biosensors With the rise of rational drug design, pharmaceutical researchers are increasingly looking for molecular explanations of a drug's effect. Biosensors can provide answers to these sorts of questions early on in the drug development process, minimizing the total number of animals needed to take a drug candidate from conception to reality. A biosensor is a biological detection system, such as an antibody-antigen or hormone-receptor complex, linked to an electronic transducer, such as a silicon chip. A biochemical change, usually in pH, is converted into a readable signal, such as a change in voltage or temperature. Molecular Devices Corp. of Menlo Park, Calif., has built a biosensor business around its light addressable potentiometric sensor (LAPS). In this device, light strikes the back of a silicon chip that is in contact with a biological sample, which lies against an insulating layer. The action of the light on the silicon generates a measurable electrical current whose magnitude is directly related to the surface potential, which reflects biological activity. The company's silicon microphysiometer, set to debut on the market in 1992, uses the LAPS setup to track the lowering of pH as metabolic rate rises (The Scientist, Jan. 22, 1990, page 24). When cells release lactic acid as a metabolic by-product, causing pH to fall, electrons are absorbed by the sensor, altering the electrical potential, which is detected by an ammeter. The silicon microphysiometer is a glass cover slip over a silicon sheet (the sensor) bearing small wells. The inner surface of the cover slip is coated with a proprietary indium-tin oxide conductive material. The space between the silicon chip and cover slip is a flow chamber for media containing cells. Adherent cells, such as fibroblasts, grow right on the cover slip. Migratory cells, such as lymphocytes, settle in the wells when the flow is halted. Laser light or a light-emitting diode from below activates the silicon. The silicon microphysiometer is fast, specific, correlates well with Draize data, and shows a time course. "Something a biosensor allows you to do that other technologies do not is to not only measure reactions of cells, but to measure their recovery," says Joan Kureczka, spokeswoman for the company. To replace a Draize test, the silicon microphysiometer determines the concentration of the suspected irritant that lowers metabolic rate of human keratinocytes by half, following a five-minute exposure. Similar tests using substitute skin products can take days. In other applications, researchers using the microphysiometer can study a cell's response to various substances, such as interleukin-2, a drug with debilitating side effects that is used to treat cancer. Back At The Bench For every in vitro test making its way into headlines and product lines, many more are in development. One farsighted project, at Washington University in St. Louis, is senior research associate Ales Prokop's work on three-dimensional organ culture. "Prokop wants to create an `artificial animal,' " says Michael Bello, executive director of the Chicago-based International Foundation for Ethical Research, which funds the work. "Critics of animal alternatives say tissue cultures cannot approximate a living organism. But Prokop is trying to link together interacting artificial organs," such as the liver and kidney, which detoxify and excrete substances. At the University of North Texas in Denton, Jen Hill Lucas, an assistant professor of biology, is using a tissue culture model of mammalian nerve cell injury to develop and screen experimental treatments for spinal cord trauma. The current experimental method involves injuring the exposed spinal cord of research animals. Lucas hopes that her method, which uses cultured fetal mouse spinal cord cells, will supplement the existing method, and ultimately lead to a reduction in the number of animals used in prescreening. Lucas explains: "We are not eliminating animal use, because you have to go from the tissue culture to in vivo testing for something that looks promising. But we can do prescreening. I personally believe animal research is necessary, but we can work on things to reduce the numbers." The quest for animal alternatives presents a terrific opportunity for academic and industrial researchers to work hand-in-hand. Consider the experiences of Robert van Buskirk, a professor of biology at the State University of New York, Binghamton, who is interested in monitoring multiple mechanisms of cytotoxicity. "Several years ago I started working on fluorescent indicator dyes that are useful in seeing what happens to cells during a toxic insult," recalls van Buskirk. "But there was no single machine to do that. I convinced Millipore [Corp.] of Bedford, Mass., to develop a spectrofluorometer that looks at fluorescent dyes in cells for doing in vitro cytotoxicity." That device, the CytoFluor, today is a top seller. It measures increases in intracellular calcium and changes in extracellular pH, both of which are indirect measures of cell toxicity. Van Buskirk is spending this summer at Millipore in collaboration with Organogenesis, where the team will put Organogenesis' Testskin living skin equivalent into the CytoFluor. "Plus, the U.S. Army just bought three CytoFluor units, and hired me as a consultant to understand how mustard gas works, and figure out an antidote," the SUNY researcher says. The search for animal alternatives is rapidly gaining momentum. In addition to federal granting agencies, funding sources for this work include a number of smaller organizations, such as the Foundation for Biomedical Research, based in Washington, D.C., and Bello's International Foundation for Ethical Research. In addition, many tests have received funding from university-based centers, such as Rockefeller University's Laboratory Animal Research Center. These centers fund projects both at their own institutions and here. Ricki Lewis is an adjunct assistant professor of biology at the State University of New York, Albany, and is the author of Life, a biology text (Dubuque, Iowa, William C. Brown Publishing , 1991).

The Search For Alternatives To Animal Testing Intensifies

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