Assessment of the genotoxic potential of ISIS 2302: a phosphorothioate oligodeoxynucleotide.

ISIS 2302, a phosphorothioate oligodeoxynucleotide with antisense activity against human ICAM-1 mRNA, was evaluated in a battery of tests to assess genotoxic potential. There was no evidence of genotoxicity in three in vitro studies performed: (i) a bacterial reverse mutation test; (ii) a chromosomal aberration test in Chinese hamster ovary cells; (iii) a mammalian cell gene mutation assay in L5187Y cells. Additionally, there was no in vivo evidence of genetic toxicity in a bone marrow micronucleus study in male and female mice. For all tests, top concentrations or doses assessed met harmonized regulatory guidelines. The cellular uptake of ISIS 2302 into target cells was confirmed using capillary gel electrophoresis and immunohistochemistry. Intracellular uptake into CHO cells, L5187Y cells, Salmonella typhimurium TA98 and bone marrow was concentration- and time-dependent. Consistent with what is known about the physical and chemical properties of phosphorothioate oligodeoxynucleotides, there was no evidence of genotoxicity in any of the assessed end-points. Furthermore, the absence of genotoxicity could not be ascribed to test system insensitivity or to an absence of exposure of the test system to ISIS 2302.

Chromosome aberration and sister chromatid exchange test results with 42 chemicals.

Forty-two chemicals were tested for their ability to induce cytogenetic change in Chinese hamster ovary cells using assays for chromosome aberrations (ABS) and sister chromatid exchanges (SCE). These chemicals were included in the National Toxicology Program's evaluation of the ability of four in vitro short-term genetic toxicity assays to distinguish between rodent carcinogens and noncarcinogens. The conclusions of this comparison are presented in Zeiger et al. [Zeiger E, Haseman JK, Shelby MD, Margolin BH, Tennant RW (1990): [Environ Molec Mutagen 16(Suppl 18): 1-14]. The in vitro cytogenetic testing was conducted at four laboratories, each using a standard protocol to evaluate coded chemicals with and without exogenous metabolic activation. Most chemicals were tested in a single laboratory; however, two chemicals, tribromomethane and p-chloroaniline, were tested at two laboratories as part of an interlaboratory comparison. Four chemicals (C.I. basic red 9 HCl, 2-mercaptobenzothiazole, oxytetracycline HCl, and rotenone) were tested for SCE in one laboratory and in a different laboratory for ABS. Tetrakis(hydroxymethyl)phosphonium sulfate was tested at one laboratory and the chloride form was tested at a different laboratory. Twenty-five of the 42 chemicals tested induced SCE. Sixteen of these also induced ABS; all chemicals that induced ABS also induced SCE. There was approximately 79% reproducibility of results in repeat tests, thus, we conclude that this protocol is effective and reproducible in detecting ABS and SCE.

Induction of sister chromatid exchanges and gene mutations in Chinese hamster ovary cells by psoralens.

Three linear psoralen compounds, 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), 3-carbethoxypsoralen (3-CP), and one angular psoralen, 5-methylangelicin (5-ANG), were tested for their ability to induce both sister chromatid exchanges (SCE) and gene mutations (hypoxanthine-guanine phosphoribosyltransferase locus) in vitro in Chinese hamster ovary cells (CHO line). All the compounds induced both SCE and mutations in the presence of UV irradiation (UVA; peak at 330-380 nm), but no increases were observed in its absence. The frequency of both responses increased with either 1) increasing concentration of compound with a fixed amount of UVA or 2) increasing amount of UVA with a fixed concentration of psoralen. Significant increases in SCE were seen for 8-MOP, 5-MOP, and 5-ANG at concentrations near 1 X 10(-6) M, whereas concentrations near 20 X 10(-6) M of 3-CP were needed before increases in SCE were observed. The induction of gene mutations followed a similar pattern; concentrations of 50-100 X 10(-6) M of 3-CP were needed to induce large increases in mutations, but much lower concentrations of 8-MOP, 5-MOP, and 5-ANG (5-10 X 10(-6) M) were sufficient to induce large increases in mutations. The ratio of induced mutations to induced SCE was similar for 8-MOP, 5-MOP, and 3-CP; that of 5-ANG was much higher, which indicated that the linear furocoumarins produce a different spectrum of DNA damage from that produced by the angular psoralen.

The effect of a tumor promotor, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on sister-chromatid exchange formation in cultured Chinese hamster cells.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) does not increase the sister-chromatid exchange (SCE) frequency in either Chinese hamster ovary (CHO) or lung (V 79) cells which are cultured in the presence of 5-bromodeoxyuridine. Moreover, TPA does not alter the induction of SCEs in CHO cells by mitomycin C during the first 3 cycles following addition of the alkylating agent. These SCE induction data do not by themselves support the hypothesis that tumor promotion by TPA depends on the enhancement of mitotic recombination.