Soft-cell Approach Cuts Animal Tests

The new in-vitro technique pioneered by Dr Amanda Hayes and her UNSW colleagues, Shahnaz Bakand and Chris Winder, directly exposes human cells to airborne toxicants and measures cytotoxic effects. The cells are grown on a porous polyester membrane inside a small diffusion chamber and then exposed to selected toxic air pollutants. After as little as one hour's exposure, they can study cell growth and metabolism, and a range of routine toxicological endpoints.

Importantly, the toxic measurements obtained by the in vitro method, such as the amount of a contaminant needed to inhibit cell growth, mirror well-established lethal values obtained from animal studies - a long-established method in toxicological studies. "In-vitro toxicity tests can improve the scientific, economic, and ethical value of research and play a significant role in the screening of toxic chemicals and the replacement of animals," Dr Hayes says.

This research earned Hayes and her colleagues the 2006 Australian Museum Voiceless Eureka Prize for Research. This prize rewards scientists for work that has reduced the use of animals or animal products in laboratory-based research, education and testing.

Many industrial and environmental air pollutants are already known to have adverse health effects on the respiratory system of workers. Increasingly, new formulations are using tiny nanoparticles and ultrafine particulates in cosmetics, pharmaceuticals and petrochemical products. Little is known about their toxicity and safety to human health but this new category of pollutants poses possible dangers, both medically and environmentally, especially if they get airborne.

Most nanoparticles have a high surface area-to-mass ratio that can make the particles very reactive or catalytic. Being so small, they may also be able to pass through cell walls in organisms, and their reactions inside the body are relatively unknown.

"These tiny new substances are the tip of a huge chemical iceberg," says Hayes, Manager of the Chemical Safety and Applied Toxicology Laboratories at the University of New South Wales.

"Worldwide, there are millions of known chemicals, of which more than 100,000 chemical compounds are in commercial use in an unknown but extremely large number of chemical mixtures.

"Continued conventional animal toxicity testing of this large number of chemicals is simply unachievable from a scientific and economic standpoint," says Dr Hayes. "It's also unethical, given that in Australia alone, more than a million dogs, cats, rabbits, sheep, cattle, pigs and mice are used each year for toxicological testing and research." This is a drop in the ocean, compared with the animal death toll in the rest of the world.

In many inhalation studies, the toxicity of airborne chemicals is tested on laboratory animals by placing them in enclosed chambers and subjecting them to increasing concentrations of the test compounds for specified times until half of the test animals are killed. One type of test commonly used where this occurs is the LD50 test, where LD stands for lethal dose.

This heavy reliance on animal data in toxicology has long been a concern of the scientific community. Predicting the biological activities of toxic chemicals in humans by using animal data always poses uncertainty due to differences between animals and humans.

In a series of published experiments, the UNSW team has demonstrated the feasibility of their in vitro technique for:

• formaldehyde, an industrial contaminant linked to human cancer;
• nitrogen dioxide, a lung irritant that causes inflammation, pulmonary oedema, and pneumonia;
• fire combustion products, including cyanide, hydrogen sulphide, and ammonia; and,
• the volatile organic compounds (VOCs) xylene and toluene, found in solvents used by the printing, painting, and petrochemical industries.

The in vitro method "opens new possibilities for toxicity testing of industrial chemicals, environmental contaminants, workplace airborne contaminants, and fire combustion products", says Dr Hayes.

The technique has several advantages over conventional tests:

• The use of human cells (as opposed to animal cells) generates data representative of direct human chemical exposure.
• A number of human cell types such as lung, skin and liver can be used to represent target organs that are more likely to be significantly exposed or affected by air pollutants. As the respiratory system is both a site of toxicity for pulmonary toxicants, and a pathway for inhaled chemicals to reach other organs, relevant airway cells and lung cells are a major focus for inhalation studies.
• The advantage of using human cell lines and cultures is that the researcher can study toxicity mechanisms at the molecular/cellular level at an earlier stage specifically for individual chemicals, depending on their site of action within the human body without using animals. For example, when assessing the toxicity of formaldehyde, which is known to have a toxic effect on the liver, liver type cells such as hepatocytes can be chosen.
• The in vitro technique is cheap and portable, so scientists can measure immediate toxicity events, rather than waiting for toxicity to be expressed as organ or organism failure many months or even years down the track from initial exposure.
• The application of in vitro methods could open new possibilities for toxicity testing of industrial chemicals, occupational and environmental contaminants, combustion products and respiratory therapeutics and lead to a better understanding of the interactions between chemical exposure and toxic effects of single chemicals and chemical mixtures.