Test Types

All three of the test types described below utilize SMPs to assess the toxic effects of materials of interest. They differ mostly in the types of reagents used in each test to control certain chemical processes which permits accessing different portions of the active enzymes in the SMP to assess toxicity. SMP are sensitive to general cellular toxicants that act by a variety of non-specific mechanisms such as disruption of membrane, membrane-protein, and protein-protein interactions in addition to the specific examples of sensitivity cited below. All tests are conducted in a standard spectrophotometer capable of reading absorbance measurements at 340nm providing results in less than 30 minutes. Aside from a personal computer for analysis of test data, no additional instrumentation is needed to conduct manual tests.

• Electron Transfer
• Reverse Electron Transfer

Electron Transfer

The Electron Transfer (ETr) assay assesses forward electron transfer through the entire electron transport chain. The test endpoint is the loss of NADH monitored as a decrease in absorbance at 340nm in a standard spectrophotometer. The ETr assay is sensitive to toxicants that inhibit Complex I, Coenzyme Q, Complex III, Cytochrome c and Complex IV. Effects are expressed through the use of the EC50; the concentration of test material resulting in a 50% reduction in the rate of loss of NADH.

Reverse Electron Transfer

The Reverse Electron Transfer (RET) assay assesses effects on electron transfer and energy coupling processes. In this test, energy derived from the hydrolysis of ATP drives electrons in the reverse direction, up the electron transport chain. The test endpoint is the production of NADH monitored as an increase in absorbance at 340nm in a standard spectrophotometer. The RET assay is sensitive to toxicants that inhibit Complex I, Complex II, Coenzyme Q, and ATPase. It is also sensitive to compounds that may damage membrane integrity, and especially to uncouplers of oxidative phosphorylation (e.g. chlorophenols). Effects in this test are also expressed through the use of the EC₅₀.