Matthews O. Bradley

DNA double-strand breaks in mammalian cells after exposure to intercalating agents

Previous work has shown that exposing mouse L1210 cells to intercalating agents such as adriamycin, ellipticine and actinomycin D results in DNA single-stranded breaks and DNA-protein crosslinks. To characterize further the interaction between these drugs and intracellular DNA we have employed a modification of the alkaline elution technique which allows the detection of DNA double-stranded breaks. Ellipticine (1.25-5.0 microgram/ml) adriamycin (0.5-3.0 microgram/ml) and actinomycin D (1.5-3.0 microgram/ml) all caused double-stranded breaks in DNA from L1210 cells following a 1 h treatment. The number of double-stranded breaks found per single strand break was highest for ellipticine, despite the fact that this is least cytotoxic of the three drugs. By comparing the single and double strand break frequency caused by radiation to that caused by ellipticine, it appears that most if not all of the drug-induced single strand breaks observed actually represent double-strand breaks. We suggest that these double-strand breaks may result from the action of an intracellular enzyme, perhaps topoisomerase, which breaks both strans in concert to relieve the topological strain caused by drug intercalation.

Relationships among cytotoxicity, lysosomal breakdown, chromosome aberrations, and DNA double-strand breaks.

Certain chemicals that are either weak or non-carcinogens had been previously shown to induce DNA single-strand breaks in rat hepatocytes, but only at cytotoxic doses. In contrast, stronger carcinogens induced DNA single-strand breaks at non-toxic doses. This report shows that the strong carcinogens and mutagens cadmium sulfate, sodium dichromate, dimethyl sulfate, and N-methyl-N-nitro-N-nitrosoguanidine all induce DNA single-strand breaks at non-toxic concentrations, but that they also induce DNA double-strand breaks at concentrations that are closely correlated with cytotoxicity. Some weak carcinogens produced DNA single- and double-strand breaks, but only at acutely cytotoxic concentrations. We suggest that the DNA double-strand breaks result from a cell-mediated process such as release of DNAase from lysosomes or other cellular compartments, that might occur during cellular response to acutely toxic damage. Experiments with N-dodecyl imidazole (NDI), a lysosomal detergent, show that lysosomal breakdown alone is only a weak inducer of DSBs, but that lysosomal breakdown in combination with prior chemical damage produced by MNNG synergistically induces DNA DSBs in BHK cells. N-Dodecyl imidazole also induces chromosomal aberrations in CHO cells at concentrations which cause cytotoxicity, cell cycle delay, and lysosomal breakdown. These results all suggest that chemical toxicity leads to limited lysosomal breakdown that induces DNA DSBs and chromosomal aberrations. Cells that have been sublethally damaged and that can repair these damages and survive could become transformed by the DNA-damaging mechanisms associated with carcinogenesis.

Significance and measurement of DNA double strand breaks in mammalian cells

Techniques for measuring DNA double strand breaks in mammalian cells are being used increasingly by researchers studying both physiological processes, such as recombination, replication, and apoptosis, as well as pathological processes, such as clastogenesis induced by ionizing radiation, chemotherapeutic drugs, and chemical toxicants. In this review we evaluate commonly used assays for measuring DNA double strand breaks, focusing on neutral filter elution and pulsed field gel electrophoresis, and explore the advantages and limitations of applying these techniques to problems of current interest in carcinogenesis and genetic toxicology.

Antisense-fos RNA causes partial reversion of the transformed phenotypes induced by the c-Ha-ras oncogene.

Several lines of evidence have suggested that c-fos may act downstream from c-Ha-ras in a growth-regulatory signal transduction pathway. We used antisense RNA to inhibit c-fos gene expression and investigated the effects of diminished c-fos expression on the phenotypes induced by the EJ c-Ha-ras oncogene in NIH 3T3 cells. Immunofluorescent staining demonstrated that the antisense RNA caused a marked reduction in the amount of c-fos protein expressed following serum stimulation. EJ cells containing antisense-fos RNA continued to overexpress ras and remained capable of proliferating in vitro. However, the antisense-fos RNA caused a partial reversion of the major transformed phenotypes of EJ cells, including a restoration of both density-dependent growth arrest and the ability to be rendered quiescent by serum deprivation, a reversion to a flat morphology, inhibition of anchorage-independent growth, and inhibition of tumorigenicity in nude mice. Our results indicate that inhibition of c-fos expression, to a level still supporting in vitro proliferation, prevents the transforming effects of the ras oncogene; they thus provide additional evidence for the participation of c-fos in ras-regulated signal transduction pathways.

A direct, highly sensitive assay for cytochrome P-450 catalyzed O-deethylation using a novel coumarin analog

The microsomal O-deethylation of a novel coumarin analog, 7-ethoxy-4-trifluoromethylcoumarin (EFC), to a fluorescent product was characterized. Results indicate that this analog provides a rapid, convenient and highly sensitive means to assay cytochrome P-450-mediated metabolism. Like microsomal 7-ethoxycoumarin (7-EC) O-deethylation, EFC O-deethylation responded to both phenobarbital was greater than that seen with 7-EC (5- to 6-fold over control after 50 mg/kg/day for 4 days in Sprague-Dawley rats compared to approximately 2-fold for 7-EC). Since the reaction was monitored by direct fluorometry of the product, any departures from linearity under a particular set of reaction conditions (e.g. with highly induced samples) were immediately apparent. In the absence of an NADPH-regenerating system, background drift was very low (less than 0.01 fluorescent units), so the sensitivity of the assay was limited primarily by that of the fluorometer employed. This makes the assay particularly useful in situations where test material is limited, e.g. when measuring activity in cultured hepatocytes. Its simplicity, reproducibility, and response to a variety of inducing agents also make it suitable for a rapid screening assay for cytochrome P-450 induction.

Cytotoxicity as a Mechanism of Carcinogenesis

Some of our previous results had shown that certain relatively weak carcinogens induced DNA single-strand breaks in rat hepatocytes but only with concomitant cytotoxicity. Stronger carcinogens usually induced breaks at relatively nontoxic concentrations. These observations led us to propose that chronic toxicity may be weakly carcinogenic through a cell-mediated mechanism without the necessity for a compound to directly attack DNA. The proposed mechanism is that one of a cells responses to sublethal toxicity is to release the contents of some, but not all, of its lysosomes. Since lysosomes contain DNA hydrolases, these could enter the nucleus and induce single- and double-strand breaks in the DNA. Such DNA damage could lead to a malignant phenotype by a variety of mechanisms in those cells that survive. Some initial support for this hypothesis comes from our observation that hypotonic shock causes single-strand breaks in the DNA of mouse L1210 cells and that these breaks can be repaired.

Rejoining of X-Ray-Induced DNA Double-Strand Breaks Declines in Unstimulated Human Lymphocytes Aging in Vivo

If DNA repair plays a major role in aging (Gensler and Bernstein, 1981; Hart et al., 1979; Little, 1976; Yielding, 1974; recently reviewed in Warner et al., 1987), within a species one would expect to see decreased efficacy of repair processes in older animals. Some data support this prediction, whereas others do not (recently reviewed in Tice and Setlow, 1985). In general, as studied in nonhuman animals, in vivo age-related DNA repair capacities differ by type of damage, by types of repair, by species, and by strain as well as by organ or tissue (Licastro and Walford, 1985; Niedermuller et al., 1985; Su et al., 1984; Tice and Setlow, 1985; Vijg, 1987).