Introduction

Many endogenous and environmental factors lead to the damage and degradation of cellular components. The presence of damaged proteins, lipids, and DNA is correlated with a variety of disease states and the aging process, demonstrating that the disruption of cellular function through the accumulation of damage products interferes with physiological function. Damage to DNA is especially harmful due to the mutations that can result if left unrepaired. Consequently, cells have developed systemic defense mechanisms that link the process for repairing DNA damage to both cell cycle regulation and controlled cell death (1,2,3,4,5,6). Cells experiencing oxidative stress conditions demonstrate an increase in the expression of the genes involved in DNA repair (7,8,9). However, once the capacity of the repair pathways is exceeded, significant problems arise. Generally, low to moderate levels of damage are countered by enzymes of the base excision repair (BAR) pathways, while high levels of damage to cellular components, including DNA, elicit an apoptotic response. High levels of DNA damage have been correlated with aging (6), type II diabetes (7,8), carcinogenesis (10,11), and auto-immune diseases (12). In each case, results have been reported that associate low levels of DNA repair enzymes, cellular anti-oxidants, and altered gene expression as consequences of oxidative stress and DNA damage. For these reasons, the ability to associate levels of DNA damage with deleterious effects is of particular interest in the medical community. Information from these measurements could potentially be developed for quantifying the level of genotoxicity induced by environmental toxins, and carcinogens, or perhaps to diagnose the early stages of disease.

Somatic human cells possess two separate genomes that are packaged and housed separately. Nuclear DNA is present as a single copy and is packaged with proteins that both condense and protect the genome. Damage to the nuclear genome is perceived to be more deleterious as there is only one copy. The circular 16.6-kb mitochondrial genome (mtDNA) is found free within the mitochondria. Due to the oxidative environment of the inner mitochondria, damage occurs frequently. This effect is partially countered by the presence of hundreds to thousands of copies of the genome in each mitochondrion. Current work has begun to use damage and mutations within mitochondrial DNA as cancer biomarkers (13). The capacity to assess damage within the two genomes and link that damage to an array of phenotypic effects would be very useful.

Potential hazardous exposures [e.g., smoking (14), pesticides (15), ultraviolet (UV)-light (16)] can be evaluated for genotoxicity and related to carcinogenesis. This association is also particularly important in determining the safety of various chemical agents and prospective therapeutic pharmaceuticals. Many techniques exist that afford the ability to identify and measure cellular DNA damage upon exposure to a suspected genotoxic agent (17). These methods include micronuclei (18), chromosome aberration test (19), unscheduled DNA synthesis (20), and the bacterial mutation test (21), each of which may be used in regulatory evaluations of potential genotoxic agents. These methods, although reliable, are often limited in their tissue application, require large sample volume, and in some cases, offer only partial data regarding primary DNA lesions. Often, the existing genotoxicity tests are limited to evaluating damage to nuclear DNA or the cell as a whole. However, in order to thoroughly understand genotoxic effects, damage to each genome should be evaluated separately.

Recent advances in cellular-based methods have resulted in the timely collection of reliable and specific data regarding levels of damage and the identity of the damage products. Antibodies developed for specific DNA damage lesions now allow for the direct measurement of those lesions within a population of exposed cells. The automation of the single-cell gel electrophoresis (comet) assay and the use of scoring software have also led to rapid data collection. Developments within the comet assay methodology have led to its acceptance and utilization by health authorities, who regard it as at least equivalent to existing techniques (i.e., micronuclei, chromosome aberration test) with regulatory acceptance (22). Under similar experimental conditions, it has also been demonstrated that the levels of DNA damage measured using the comet assay are comparable with those observed using high-performance liquid chromatography (HPLC) analyses (23).