Uses And Interpretation Of Data

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The uses of the in vitro micronucleus assay are too numerous to review, but recent examples are described briefly, below. One major use of the assay, not discussed here because it is beyond the scope of this chapter, is in assessing environmental and occupationally induced chromosome damage in vivo in humans.

A. Chemical-Specific Investigations

The in vitro micronucleus assay is used to study the genotoxicity of specific chemicals of interest. These type of studies often use the standard methods and cell types described above. However, some reports have shown how the in vitro micronucleus assay can be modified to address specific questions. Saranko and Recio (61) compared the ability of 1,2:3,4-diepoxybutane (DEB) (a metabolite of 1,3 butadiene, a genotoxic carcinogen) to induce micronuclei with mutations in Big Blue Rat 2 lambda/lacI transgenic fibroblasts. These studies were conducted to help define the role of genotoxicity of this metabolite in the overall carcino-genicity of 1,3 butadiene.

Another recent example of a chemical-specific study was reported for 1,4-dioxane (a presumed nongenotoxic rodent carcinogen) (62). The in vitro micro-nucleus assay was used in a battery of standard genotoxicity tests to confirm that 1,4-dioxane was not genotoxic in vitro, although a positive result in a liver micronucleus assay was obtained.

Ong et al (63) reported the induction of micronucleated and multinucleated cells by glass fibers of various types and sizes. The presence of kinetochore-positive staining in the micronuclei and the induction of multinucleated cells was concluded to indicate that these fibers induced aneuploidy and inhibited cytokinesis.

B. Structure Activity Relationships

Snyder (56) reported the results of the in vitro micronucleus assay to study a series of antihistamines. Results of this work, along with other mechanistic data, suggest that the genotoxicity of various antihistamines is caused by DNA intercalation, which was not expected based on the structure of these drugs. From a structure activity relationship (SAR) standpoint, Snyder was able to identify the need for a dimethylamino substituent for the genotoxic activity of these intercalating antihistamines (56).

Because the in vitro micronucleus assay detects both clastogenic and aneu-genic activity, Tafazoli and Krisch-Volders (64) and Tafazoli et al (65) used the in vitro micronucleus assay in human lymphocytes to compare the genotoxicity of various chlorinated hydrocarbons. In these studies, the results of the micro-nucleus assay were compared with the alkaline single cell gel electrophoresis assay (Comet assay).

Quinolones are a class of antibacterial compounds that inhibit bacterial DNA gyrase and can cross-react with mammalian topoisomerase, which results in genotoxicity. The in vitro micronucleus assay has been used by different labo ratories to investigate the SAR of different quinolone compounds to aid in the drug development process (66-68). Recently, Snyder and Cooper (69) reported the adaptation of the in vitro micronucleus assay for assessing the photogeno-toxicity of structurally related fluoroquinolones.

The in vitro micronucleus assay has been used as part of a multiendpoint assessment. Again, because the in vitro micronucleus assay detects aneuploidy-inducing activity, Pfeiffer et al [70] used the in vitro micronucleus assay in V79 cells to study the aneuploidy-inducing potential of bisphenols. As part of the multiendpoints assessment, they measured disruption of cytoplasmic microtu-bules, disruption of the mitotic spindle, and induction of both metaphase arrest and micronuclei by various bisphenols.

C. Correlation to Other Endpoints

The transformation of primary Syrian hamster embryo (SHE) cells in vitro has been shown to be a useful tool for the identification of both genotoxic and non-genotoxic rodent carcinogens (71). One way to help determine whether a positive response in the SHE cell transformation assay is caused by a genotoxic or non-genotoxic mechanism is to measure micronucleus induction in SHE cells. As reviewed by Tsutsui and Barrett (72), an assessment of micronuclei in SHE cells treated with various estrogens has been useful in understanding the role of geno-toxicity, particularly aneuploidy, in the transforming ability of these compounds. Gibson et al (73) reported a good agreement between the SHE cell micronucleus assay and SHE cell transformation assay for mutagenic chemicals, but a number of chemicals were negative in the SHE micronucleus assay that induced cell transformation, as would be expected because transformation can occur via a nongenotoxic mechanism.

Eckert et al (18) studied the relationship between induction of micronuclei containing chromosome 11 (containing the tk gene) and mutations in the L5178Y mouse lymphoma assay. Although micronuclei containing chromosome 11 were induced by various aneugens, stable, viable mutants were not recovered.

Van Goethem et al (74) compared induction of micronuclei in vitro and the alkaline single cell gel electrophoresis (Comet) assay with various cobalt compounds. Their results demonstrate how these two methods can be used to investigate more directly the relationship between induced DNA strand breaks and formation of micronuclei.

D. Thresholds

To obtain the statistical power needed to detect small differences above background and establish a no-effect-level, or threshold, for a genotoxic event requires the analysis of a large number of cells. Because the analysis of micronuclei is relatively easy and fast, it is practical for the study of thresholds for genotoxins. Elhajouji et al (75, 76) and Marshall et al (77) used the in vitro micronucleus assay in human lymphocytes to determine practical thresholds for a variety of spindle inhibitors. In these studies, different endpoints were measured to help establish a threshold, including the general induction of micronuclei (75-77), the induction of centromere-positive micronuclei (indicative of micro-nuclei containing whole chromosomes) that had been collected by flow cytome-try (75), and the induction of nondisjunction of specific human chromosomes in the interphase nuclei of cytokinesis-blocked cells (76, 77). In addition to providing critical data to support the existence of thresholds for spindle inhibitors, these studies resulted in an important observation that the measure of micronuclei (that result from chromosome loss) is a less sensitive measure of aneuploidy, compared with the measurement of nondisjunction (the irregular distribution of chromosomes to the daughter nuclei).

E. Affect of Different Genotypes

The in vitro micronucleus assay provides an easy method for assessing the influence of different genotypes on induction of genotoxicity. These studies involve genetically engineered cell lines, as well as human and rodent genetic variants. To investigate the effect of metabolism on genotoxicity of nitroarenes and aromatic amines, sublines of Chinese hamster cells stably expressing human NAT1 or NAT2 N-acetyltransferases or Salmonella typhimurium O-acetyltrans-ferase were compared for sensitivity to micronucleus induction (78). Cells expressing human NAT2 N-acetyltransferase had the highest sensitivity to chromosome damage induced by nitroarenes. Norppa (79) discussed how the in vitro micronucleus assay (among other cytogenetic methods) can be used to study the effect of human polymorphisms, such as in the detoxification enzyme glutathione S-transferase.

F. Biomarker of Human Cancer Predisposition, Treatment Outcome

Because increased sensitivity to radiation-induced chromosome damage is common in people predisposed to cancer, the in vitro micronucleus test has been investigated as a tool for identifying increased carcinogenic risk in humans. Scott et al (80, 81) reported increased radiation-induced micronuclei in peripheral blood lymphocytes from breast cancer patients. They proposed that this assay may be useful for identifying women with a predisposition for breast cancer. The use of the in vitro micronucleus assay for determining clinical outcome of various cancers was reported by Shibamoto et al (82). Because of the potential power of this method as a biomarker of human disease and treatment, a collaborative project was initiated to define the assay better (35).

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