January 2006
With the completion of the Human Genome Project, we have now truly entered the exciting era of post-genomics biology. Several new scientific disciplines have emerged of which metabolomics holds significant promise for the understanding and diagnosis of diseases both in humans and wildlife. This introduction to the new field of metabolomics will describe several applications of this approach for monitoring the health of organisms in the environment.
The inside of a Californian red abalone shell, Haliotis rufescens (compare size to the coin in photo). The shellfish is susceptible to a disease called withering syndrome. Source: Wikimedia Commons.
Introduction to metabolomics
Metabolomics is the study of all the naturally occurring small molecules, called metabolites, in biological samples such as cells, biofluids, or tissues. These small molecules are the products of metabolism and include, for example, sugars (or carbohydrates), fats (or lipids), and amino acids. The collection of all the metabolites within a cell is called the metabolome. Scientists have started to characterize the metabolome in a quest to better understand and diagnose disease.
- Metabolomics incorporates the use of bioinformatics, the application of computer and statistical techniques to the understanding and management of biological information, to search for unique patterns of metabolites that are indicative of a particular disease.
- Metabolomics is a multidisciplinary approach involving biologists, computer scientists, and analytical chemists. The tools used to measure the metabolites are more commonly associated with chemistry laboratories and include nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry.
The advantage of metabolomics for disease diagnosis, whether in humans or wildlife, stems from the fact that this approach measures the phenotype of an organism, the biological characteristics of an organism that result from the interaction of its genetic make-up with the environment. When an organism becomes diseased or stressed, thus triggering specific molecular changes, the phenotype becomes altered. This change can then, in principle, be measured using metabolomics.
Current applications in human disease diagnosis
For many years doctors have been measuring specific metabolites in a patient’s blood or urine to diagnose particular diseases. Perhaps the most familiar is the measurement of glucose to diagnose diabetes. Metabolomics is opening up new horizons as hundreds of metabolites can be measured rapidly and simultaneously, providing a much more comprehensive assessment of a patient’s health status. Recently, notable applications of metabolomics in the study of human diseases have begun to emerge:
- Detection of the presence and severity of coronary heart disease using NMR-based metabolomics.1 This noninvasive approach identified the disease from human serum samples and in the future could reduce the use of angiography, which is highly invasive.
- Prediction of the clinical outcome of a sudden hemorrhage of a blood vessel over the surface of the brain (termed subarachnoid hemorrhage), by means of metabolomics analysis of cerebral spinal fluid.2
- Classification of patients with progressive neurological diseases (e.g., amyotrophic lateral sclerosis, in which loss of nerve cells produces muscle paralysis) into clinically relevant groups on the basis of metabolite profiles in serum samples.3
Metabolomics in the environmental sciences
Researchers have been developing and applying these methods to study the effects of both diseases and chemicals on wildlife species, or “environmental organisms.” This is a particularly important area of science for several reasons, ranging from concern over the health of the environment to maximizing profits for the aquaculture industry. Environmental metabolomics may prove of major benefit in a variety of ways:
Environmental monitoring using so-called “sentinel species” of vertebrate and invertebrate animals. Many organizations, typically government related, monitor the prevalence of diseases in certain species of wildlife as indicators of the health of the environment. For example, within the United Kingdom the National Marine Monitoring Program collects several fish species to assess the effects of disease, pollutants, and other stressors such as climate change on fish stocks and biodiversity in the aquatic environment.
Chemical risk assessment of pharmaceuticals, pesticides, and other household and industrial chemicals. Prior to the use of any new chemicals in society, the company that has developed and manufactured the chemical must assess the risk posed to wildlife and the environment. Only if a new chemical poses minimal harm can it be licensed and sold.
- Maintenance of healthy stocks of animals in the aquaculture industry, including fish and invertebrates. As with any type of intensive farming, rearing large numbers of animals in close proximity can drastically increase the occurrence and spread of diseases. Maintaining healthy animals is important for both animal welfare and productivity.
Identification of cancer in marine flatfish
Metabolomics and proteomics, the study of thousands of proteins simultaneously, have been used to study liver cancer in a marine flatfish species called dab (Limanda limanda).4 Scientists had noted high levels of tumours in up to 14 percent of the fish collected from the open sea and estuaries around the United Kingdom. It was hypothesised that metabolomics and proteomics could identify differences between healthy and diseased dab livers, and that these differences, or biomarkers, could be used to rapidly diagnose liver cancer in the future.
Initial studies using mass spectrometry did indeed find molecular differences between healthy and diseased livers, although the exact metabolites remain unidentified. The goal of the investigation is to identify the specific causes within the environment that may be responsible for the disease. Potential causes include chemical pollutants that are ingested by the bottom-feeding dab or biological factors such as bacteria or viruses.
Chemical risk assessment in fish, mammals, and earthworms
A number of research groups have been developing and using metabolomics to study the effects of chemicals on organisms in the environment. In addition to the work on aquatic organisms, several studies on terrestrial invertebrates have been conducted, and a limited number of studies on terrestrial mammals have been reported. The advantage of metabolomics over traditional approaches for assessing the effects of chemical toxicity is that earlier methods tend to measure only a small number of responses. With metabolomics, hundreds of metabolites can be monitored simultaneously, providing a much more comprehensive snapshot of the effects that a particular chemical has on a living organism.
- Studies were conducted using Japanese medaka (Oryzias latipes), a species that is widely used in toxicity testing, to investigate the effects of trichloroethylene, an environmental pollutant, and the pesticide dinoseb on the development of fish embryos.5,6
- Other metabolomics studies have identified biomarker patterns in earthworms (Eisenia veneta) following exposure to pollutants such as a nitrophenol7 and fluorinated anilines.8
- The effects of arsenic, a common environmental contaminant, on kidney metabolism in the bank vole (Clethrionomys glareolus) have also been investigated using NMR-based metabolomics.9
Monitoring withering syndrome in California red abalone
Red abalone (Haliotis rufescens), an important shellfish species that lives along the Pacific Coast of the United States, is susceptible to a disease called withering syndrome. This fatal disease is caused by a bacterial infection and is known to have decimated more than 90 percent of the related black abalone (Haliotis cracherodii) population in southern California.
- The potential impact of withering disease on the aquaculture industry prompted the use of metabolomics to identify and measure multiple biomarkers associated with the disease. Using NMR-based metabolomics, characteristic fingerprints of metabolites were detected in the foot muscle, digestive gland, and hemolymph (blood) in diseased abalone that were different from those in healthy animals.10
Building upon this research, scientists have since investigated the influence of food availability, temperature, and bacterial infection on the health status of the red abalone.11 They have shown that withering syndrome depends on bacterial infection, and that metabolomics correlate well with the more painstaking inspection of the tissue under a microscope.
Furthermore, scientists confirmed that a particular ratio of two metabolites, glucose and homarine, in foot muscle serves as a biomarker for distinguishing diseased animals from both healthy and starved abalone.
Metabolomics have also been used to determine whether treatment with an antibiotic, oxytetracycline, can reverse the effects of withering syndrome. The results from this study are still pending (for further information, check the websites listed at the end of this article).
Future developments in environmental metabolomics
Although much progress has been made in environmental metabolomics in the past few years, researchers have only scratched the surface in terms of potential applications. This is partly because this approach is still technically complicated, limiting its widespread introduction into environmental laboratories. Indeed, considerable work still remains in developing the chemical and computational technologies that underpin this science. As the technology advances, we will better realize and exploit the advantages of metabolomics for studying disease and toxicity in wildlife. The point is to be able to diagnose the health of organisms using metabolomics analyses of minute blood samples, and then to relate these measurements on individuals to the overall health of the environment, particularly the impacts of pollution, climate change, and other manmade stressors.
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