Halogenated squaraine dyes 1 and 2 possess favorable photophysical and in vitro photobiological properties that make these new class of molecules interesting for photodynamic therapeutic applications. For a better understanding of the mechanism of their photobiological activity, we have analyzed the DNA damage and the cytotoxicity induced by these photosensitizers in mammalian cells and cell-free systems in the presence and absence of various additives and scavengers. Both photoactivated squaraines were found to be similar efficient in inducing single-strand breaks (SSB) in cell-free DNA when compared with the cellular DNA. Superoxide dismutase and catalase did not show any influence. However, the presence of tert-butanol and glutathione inhibited the formation of the DNA SSB, indicating an indirect (possibly squaraine radical mediated) mechanism under cell-free conditions. Replacing H2O in the buffer by D2O resulted in a five- to six-fold increase in the number of the SSB in cell-free DNA and a significant enhancement of the photocytotoxicity in mouse lymphoma cells. The results demonstrate that singlet oxygen is the major reactive species under cell-free and cellular conditions and confirm that squaraine-based sensitizers 1 and 2 can have potential applications in photodynamic therapy.

Halogenated squaraine dyes are characterized by long wavelength absorption (>600 nm) and high triplet yields and therefore represent new types of photosensitizers that could be useful for photodynamic therapy. We have analyzed the cytotoxicity and genotoxicity of the bromo derivative 1, the iodo derivative 2 and the corresponding nonhalogenated dye 3 in the absence and presence of visible light. At concentrations of 1-2 microM, 1 and 2 reduced the cloning efficiency of AS52 Chinese hamster ovary cells to less than 1% under conditions that were well tolerated in the dark. Similarly, the proliferation of L5178Y mouse lymphoma cells was inhibited by photoexcited 1 and 2 with high selectivity. The squaraine 3 was much less efficient. Both 1 and 2 induced only few mutations in the gpt locus of the AS52 cells in the presence of light and were not mutagenic in the dark. No mutagenicity with and without irradiation was observed in Salmonella typhimurium TA100 and TA2638. However, both 1 and 2 plus light increased the frequency of micronuclei in AS52 cells. The results indicate that halogenated squaraines exhibit photobiological properties in vitro that are favorable for photodynamic therapeutical applications.

The oxidative DNA damage induced by the polar photosensitizer Ro19-8022 in the presence of light was studied and correlated with the associated mutagenicity. Both in isolated DNA and AS52 Chinese hamster ovary cells, photoexcited Ro19-8022 gave rise to a DNA damage profile that was similar to that caused by singlet oxygen: base modifications sensitive to the repair endonuclease Fpg protein, which according to high-performance liquid chromatography (HPLC) analysis were predominantly 8-hydroxyguanine (8-oxoG) residues, were generated in much higher yield than single-strand breaks, sites of base loss (AP sites) and oxidative pyrimidine modifications sensitive to endonuclease III. Fifty percent of the Fpg-sensitive modifications were repaired within 2 h. Under conditions that induced 10 Fpg-sensitive modifications per 10(6) bp (six 8-oxoG residues per 10(6) bp), approximately 60 mutations per 10(6) cells were induced in the gpt locus of the AS52 cells. A rather similar mutation frequency was observed when a plasmid carrying the gpt gene was exposed to Ro19-8022 plus light under cell-free conditions and subsequently replicated in bacteria. Sequence analysis revealed that GC-->TA and GC-->CG transversions accounted for 90% of the base substitutions. A significant generation of micronuclei was detectable in AS52 cells exposed to the photosensitizer plus light as well.

Chinese hamster ovary cell lines (AA8 and AS52) were stably transfected to overexpress hOgg1 protein, the human DNA repair glycosylase for 7,8-dihydro-8-oxoguanine (8-oxoG). In the transfectants, the repair rate of 8-oxoG residues induced by either potassium bromate or the photosensitizer [R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)-carbo nyl ]-2-pyrrolidinemethanolplus light was up to 3-fold more rapid than in the parental cells. However, the improved repair had little effect on the mutagenicity of potassium bromate in the guanine phosphoribosyl transferase (gpt) locus of the OGG1-transfected AS52 cells. The steady-state (background) levels of DNA base modifications sensitive to Fpg protein, which include 8-oxoG, in cells not exposed to a damaging agent were not reduced by the overexpression of Ogg1 protein. Moreover, the spontaneous mutation rates in the gpt locus were similar in OGG1-transformed and vector-only-transformed cells. The results demonstrate the potential of Ogg1 protein to remove its substrate modifications from most of the chromosomal DNA. They indicate, on the other hand, that the Ogg1 protein alone may not be rate limiting for the repair of the residual substrate modifications observed in cells under normal growth conditions.

Loss of heterozygosity is one genetic change observed in many tumours, We do not know whether the loss of chromosomal material through micronucleus formation is a viable mechanism associated with, and possibly leading to, genetic disease, Previously, we treated L5178Y mouse lymphoma cells with four aneugens, Although these aneugens induced micronuclei containing predominantly whole chromosomes, they did not induce mutations at Tk1, the selectable gene, under the same non-toxic conditions in which they induced micronuclei, This suggested that the induction of micronuclei containing whole chromosomes was not an early event leading to phenotypically expressed mutations in these cells under the conditions used, However, it is possible that chromosome 11, on which Tk1 resides, may be under-represented in the micronucleus population, To find out the frequency of induction of micronuclei containing chromosome 11, we applied fluorescence in situ hybridization using a chromosome 11 paint to micronuclei induced by colcemid and vinblastine, We found that the numbers of micronuclei containing chromosome 11 are more than sufficient to be detectable as mutations if these micronuclei lead to viable mutants, We conclude that the formation of micronuclei containing whole chromosomes does not lead to viable, dividing mutants in this system.

In mammalian DNA, 3%–5% of cytosine residues are present as 5-methylcytosine (Holiday and Grigg 1993). This methylation is involved in several epigenetic processes, including genomic imprinting and gene expression (Tilghman 1993). DNA hypomethylation is one of the possible causes and one of the consequences of mammalian neoplasia and also occurs in cultured cells after treatment with certain carcinogenic drugs (Spruck et al. 1993). Changes in DNA methylation can affect DNA conformation (Bird 1992).

The carcinogenic endogenous hormone beta-oestradiol induces genotoxic effects in short-term mammalian tests. In this study we investigated micronucleus induction in V79 cells. The early induction of primarily kinetochore positive micronuclei indicated a mechanism involving mitotic disturbances. Metaphase arrangements that showed displaced chromosomes suggested that these chromosomes may be enclosed in micronuclei. However, observation of live cells revealed that most of the displaced chromosomes reintegrated. In some of these cells chromatid separation at anaphase was impaired and a small chromatin fragment was separated from the remaining chromatin.

This study was designed to investigate a previously unidentified potential mechanism for mutation induction as well as to clarify a biological consequence of micronuclei with mutation induction as measured by trifluorothymidine (TFT) resistance in mouse L5178Y cells using four aneugens: colcemid, diethylstilbestrol, griseofulvin and vinblastine. All four compounds induced micronuclei which appeared in the first cell cycle after treatment. More than 85% of the micronuclei induced by each compound stained positive for the presence of kinetochores implying that the micronuclei contained whole chromosomes. However, these same compounds were unable to induce TFT resistance under three different treatment regimes. We concluded that these compounds, under conditions where they induce primarily kinetochore positive micronuclei, were not able to induce mutations. Thus, the induction of micronuclei containing whole chromosomes harboring a selectable gene is not an early event leading to mutations in these cells.

The structure of the (1 x 2) ''streaky phase'' formed upon adsorption of deuterium on Ni(110) was studied using He scattering. The presence of a pronounced rainbow pattern in the in-plane-spectra observed with the beam incident along [001] signals unambiguously that the ''streaky phase'' is of the missing-row structure type. The corrugation of the (I X 2) troughs could be determined as approximately 1.55 angstrom, which represents an increase of approximately 0.3 angstrom with respect to the estimated corrugation of a clean Ni-missing-row surface; this observation suggests that the deuterium atoms occupy sites near the ridges of the missing-row troughs. From out-of-plane measurements of the remnant (2 x 1) phase, the fraction of unreconstructed Ni domains coexisting with the missing-row structure has been estimated as 10%.

Some chromosomes in transformed rat cells and somatic cell hybrids fail to display the presence of kinetochore proteins as detected by antikinetochore antibodies. Such chromosomes (K- chromosomes) may constitute a novel mechanism for the genesis of aneuploidy. We have analyzed primary, immortalized and malignant mammalian cells for the presence of kinetochore proteins and micronuclei. Our results suggest a correlation of the K- chromosome and micronucleus frequency with the variability in chromosome number. Upon in situ hybridization with the minor satellite and alpha satellite sequences some K- chromosomes showed a signal. This indicates that the observed lack of kinetochores is not necessarily due to a lack of centromeric DNA. We conclude that dislocated K- chromosomes may become incorporated into micronuclei which are prone to loss. Such events would be associated with the generation of aneuploidy.