The Further Development of a Mammalian DNA Alkaline Unwinding Bioassay With Potential Application to Hazard Identification for Contaminants from Environmental Samples

Abstract

Recently, we have detailed a DNA alkaline unwinding assay (DAUA) that can be used to rapidly measure chemically induced strand breaks in mammalian cells (Daniel et al., 1985). In this paper we present further development of this assay, including: (1) studies on the relationship between DNA adducts and DNA strand breaks; (2) evaluation of the role of cytotoxicity in DNA strand breaks; and (3) application of the DAUA to cell preparations from the liver of mice dosed with methylating agents. The level of DNA adducts produced in human CCRF-CEM cells by treatment with benzo(a)pyrene diol-epoxide (BPDE), N-acetoxy-2-acetyl aminofluorene (AAAF), and various methylating agents was linear with concentration over several orders of magnitude. Likewise, the level of strand breaks increased with the concentration over the same dose range. The strand breaks/adduct ratio ranged from 0.05 for the methyl adducts to 0.001 for the BPDE adducts. Using these values and the inherent sensitivity of the DAUA (circa 100 to 1000 breaks/cell), (Daniel et al., 1985), the ability of the assay to detect DNA damage induced by various classes of chemical carcinogens can be calculated. The DAUA appears to be useful for assessing the relative potency of various environmental genotoxic effects on mammalian cells. In addition, it can be conducted on cells isolated from target organs of whole animals.

Analysis of DNA strand breaks induced in rodent liver in vivo, hepatocytes in primary culture, and a human cell line by chlorinated acetic acids and chlorinated acetaldehydes

“…The direct measurement of DNA damage is a useful parameter for assessing the genotoxicity of environmental contaminants [Kohn, 1983;Daniel et al, 1985Daniel et al, , 1989Garberg et al, 1988;Chang et al, 1991). One type of DNA damage, DNA strand breakage (DNA SB), often occurs as an early consequence of the interaction between genotoxic agent and mammalian DNA.…”

“…CAA was a potent inhibitor of DNA synthesis [Kandala et al, 19901, formed interstrand cross-linkages with DNA in vitro [Spengler and Singer, 19881 and modified DNA conformation [Singhal and Landes, 19881. It was shown to be mutagenic in bacteria [McCann et al, 19751 and Chinese hamster cells [Huberman et al, 19751 and induced aneuploidy in Asperigilfus [Crebelli et al, 19901. The direct measurement of DNA damage is a useful parameter for assessing the genotoxicity of environmental contaminants [Kohn, 1983;Daniel et al, 1985Daniel et al, , 1989Garberg et al, 1988;Chang et al, 1991). One type of DNA damage, DNA strand breakage (DNA SB), often occurs as an early consequence of the interaction between genotoxic agent and mammalian DNA.…”

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“…MMS induced the most DNA SB at 50,020 SB/cell/μM (56.01% damage), whereas TBM was the most potent THM, inducing 16,800 and 34,030 SB/cell/μM (24.12 and 42.79% damage) at 5 and 10 mM, respectively. The numbers given for DNA SB induced per cell were calculated as described by [16,17]. …”

“…The relationship between the F-value and the number of DNA SB induced per cell (Ni) as a function of concentration or the test chemical has been determined previously for CCRF-CEM cells [16,17]. The number of DNA SB induced (Ni) per cell was calculated as Ni = -6.1 × 10 4 [ln (F T /F O )], where Ni is the number of DNA SB per cell induced, F T and F O are the F values for the treated cells and control cells, respectively.…”

Assessing Genotoxicity of Ten Different Engineered Nanomaterials by the Novel Semi-Automated FADU Assay and the Alkaline Comet Assay

“…A decrease in SybrGreen ® fluorescence intensity in the cell lysates indicates an increase in progressive DNA unwinding and, thus, represents a greater number of DNA strand breaks. The applicability of this method has been demonstrated for the detection of mutagen-induced DNA strand breaks [20,31], the repair of UV-induced DNA strand breaks [32], environmental genotoxic effects [33] and ZnO nanoparticles [34]. The novel semi-automated version of this assay enables the fast assessment of DNA breakage in a 96-well-plate format and the integration of different control samples for ENM interference detection.…”