Production by lithocholic acid of DNA strand breaks in L1210 cells.

Bile acids have been reported to promote colon cancer cells in mice treated with different carcinogens. In this study, we investigated the effects of lithocholic acid on the DNA of mouse lymphoblastoma L1210 cells. Incubation of L1210 cells with lithocholic acid (2.5 X 10(-4) M) at 37 degrees for 30 min and for 1 hr resulted in the appearance of single-strand breaks in the DNA. This was demonstrated by sedimentation of nucleoids in neutral sucrose gradients and by alkaline elution. The DNA damage was repaired upon incubation of the cells in fresh medium lacking lithocholic acid. These results suggest that DNA repair efficiency is an important function for the population of cells which are constantly exposed to low concentrations of lithocholic acid. The presence of even a low level of persistent damage could lead to significant biological consequences including mutations and the induction of error-prone repair processes.

Intracellular binding of ethidium studied by photoaffinity labeling in vivo.

The azide analog of 14C-labeled ethidium bromide was mixed with yeast cells and when photolyzed by visible light, formed covalent complexes with all yeast cell organelles. The 14C counts were found in DNA, RNA and protein of yeast subcellular fractions, illustrating the complexity of binding of a drug which appears highly specific in its actions.

Binding of ethidium monoazide to the chromatin in human lymphocytes.

The azide analog of [14C]ethidium bromide was mixed with lymphocytes and photolyzed with visible light. The distribution of azide in the chromatin fraction was found to be 55% in DNA, 28% in protein and 16% in RNA. Label in the DNA portion was found to be almost exclusively in the region digestible with micrococcal nuclease. The parent compound, ethidium bromide, competed with azide for binding sites, illustrating that the azide analog mimics the action of ethidium bromide.

Synthesis, separation and characterization of the mono- and diazide analogs of ethidium bromide.

Ethidium bromide is used to characterize nucleic acid secondary and tertiary structural properties and the biological consequences of drug interactions. The mono- and diazido analogs of ethidium have proven valuable as photoaffinity probes in chemical and biological studies on nucleic acids, since they render the ethidium-nucleic acid interaction covalent. Although both of these compounds have been synthesized previously, the published synthesis procedure for the monoazide is inadeqlate since a major portion of the product has been identified as the diazide analog. This lack of purity severely limits the usefulness for nucleic acid research. The procedure presented here for the synthesis, separation, purification and crystallization of these analogs should provide the quantities and quality of these important reagents needed to perform a variety of chemical and biological experiments.

Direct in vitro photoaffinity labeling of DNA with daunorubicin, adriamycin, and rubidazone.

Irradiation by daylight fluorescent lamps, of a physical complex of duplex DNA with either daunorubicin, adriamycin, or rubidazone produced covalent adducts to DNA. The photoincorporated drug could not be extracted by phenol extraction which removed 99% of the non-photolyzed drug from its physical complex with DNA. The photoadduct was also stable to dialysis, Mg2+ addition, column chromatography, and thermal denaturation and alkali treatment of the DNA. The photoinduced adduct was proportional to the amount of incident irradiation, and the amount of DNA present, and as much as 30--45% of the drug which was physically associated could be photoincorporated. The drugs were not incorporated extensively into single-stranded DNA which lacks the ability to bind these antitumor agents by intercalation. Although the photochemical mechanisms of photoaffinity labeling DNA with these antineoplastic agents are unknown, this approach may prove to be useful for trageting their cellular sites of actions.

Observations on epidermal exsorption in mice following injections of procion dyes and ethidium bromide and topically applied dimethyl sulfoxide.

Back skin of hairless mice and external ears of CD-1 white mice were used to study the details of epidermal exsorption . Ethidium bromide, a DNA ligand, and two dichlorotriazinyl (procion) dyes were injected, i.v. or i.p. Migration patterns from the skin vasculature into the epidermis were observed by fluorescence microscopy and microspectrofluorometry . Topically applied DMSO greatly enhanced the exsorption process and produced intensely labeled epidermis. Ethidium bromide reacted primarily with nuclear DNA of living cells while the procion dyes tended to migrate intercellularly to label the stratum corneum. External ears of white mice treated topically with DMSO showed a pattern of labeling which included intense fluorescence of the cartilage and perichondrium as well as the ear epidermis.

Involvement of deoxyribonuclease activity in the differential sedimentation rates of nucleoids from non-transformed and transformed mouse embryo fibroblasts.

Lysis of non-transformed confluent C3H10T1/2C18 mouse embryo fibroblasts in the presence of detergents, high concentrations of salt and EDTA on top of neutral sucrose gradients revealed a reduced sedimentation rate of the resulting nucleoids from these cells compared to those from exponentially growing non-transformed cells or from transformed cells. Exposure of confluent cells to 1000 rads of X-ray had no effect on this rate of nucleoid sedimentation; and ethidium bromide titration and alkaline sucrose analysis suggested the presence of discontinuities in the DNA. An endonucleolytic activity could be extracted from nuclei of these cells with 0.5 M NaCl, indicating a very tight association with the chromatin. Such an enzyme in non-transformed confluent cells may account for the differences in nucleoid structure and may be related to changes in cell function with normal arrest of cell growth. There was no growth-phase effect on the properties of nucleoids from transformed cells.

Photosensitive cataractogens, chlorpromazine and methoxypsoralen, cause DNA repair synthesis in lens epithelial cells.

Autoradiographic techniques showed that photoactivation of methoxypsoralen opr chlorpomazine caused diffuse nuclear labeling of lens epithelial cells by thymidine. Chlorpromazine and light also induced thymidine incorporation into lymphocytes, whereas light or drug alone did not cause unscheduled DNA synthesis.

Primary and secondary risk factors for birth defects.

Birth defects may be inherited in the germ line or may result primarily from a wide spectrum of predictable physical, chemical, and infectious processes that can operate in the mother, the father, or in the zygote. The systematic consideration of these mechanisms can lead to a fresh awareness of risk and possible strategies toward recognizing and avoiding such risks. Birth defects also depend heavily on secondary factors that may even be of greater concern than any single primary insult because they may simultaneously affect the consequences of more than one primary exposure. Under the influence of secondary factors, the frequency, timing, and intensity of developmental deficiencies can be quite varied. It is particularly interesting that expression can be delayed until quite late in life, and deficiencies may occur or be expressed only in response to the appropriate environmental stress or functional demand. Any discussion of teratogenic mechanisms, therefore, is not complete without taking into account the important concept of co-teratogenesis, or the operation of secondary risk mechanisms. The principle of secondary risk or co-teratogenesis has been demonstrated by means of enhancement of radiation-induced terata by the administration of drugs that inhibit DNA repair. An example of late-onset expression of prenatal damage was illustrated with postnatal retinal degeneration occurring after prenatal damage to the developing retina. It is suggested that a systematic consideration of primary and secondary risk mechanisms can lead to a better understanding of the problem of birth defects.

Focal cerebellar dystrophy caused by transplacental administration of methylnitrosourea.

Methylnitrosourea (MNU), 20 mg/kg, was given IP to CD-1 mic on day 16 of pregnancy and the offspring examined at 3 and 5 weeks of age. In addition to a general reduction in brain size in all specimens, there were focal alterations in cerebellar architecture. Specifically, the granule cells of the anterior lobe and vermis were reduced in number and ectopic in localization. There were concomitant changes in the localization of the Purkinje cells suggesting changes in migration influences. These experiments used a short-lived (15 minute), direct-acting DNA alkylating agent to produce focal cerebellar damage. MNU therefore, appears to be a promising tool for examining regional developmental abnormalities in the central nervous system.

Effect of verapamil on antitrypanosomal activity of drugs in mice.

The effects of verapamil on drug responses of Trypanosoma brucei brucei were studied to determine whether drug resistance of this organism could be related to expression of a drug resistance gene as has been described for drug-resistant cancer cells and malaria. Concomitant administration of verapamil during treatment of two different strains of the parasite with ethidium or berenil resulted in enhancement of the drug effect as shown by increased formation of dyskinetoplastic organisms, increased rates of clearing of the parasites from the blood, and by enhanced survival of infected mice. Verapamil treatment was associated with increased intracellular accumulation of drug, as shown by fluorescence of cells exposed to ethidium or DAPI, a fluorescent surrogate for berenil. These results suggest the importance of exploring the expression of the multiple drug-resistance gene in this series of parasites.

Use of drug-specific antibodies to identify ethidium adducts produced in Trypanosoma brucei by photoaffinity labeling.

A photoreactive azido analog of the trypanocide ethidium bromide, 3-amino-8-azido-5-ethyl-6-phenylphenanthridinium chloride, attached covalently to calf thymus DNA (CT DNA) by photoaffinity labeling, was used to generate antibodies for the drug analog. The specificity of the antiserum was tested using enzyme-linked immunoadsorbant assays (ELISA) against immobilized antigen (photoaffinity labeled DNA) and by both the avidin-biotin peroxidase reaction and indirect immunofluorescence performed on smears of drug treated trypanosomes. The reaction of the antiserum with the covalently bound drug adduct was diminished effectively by prior incubation with an excess of ethidium monoazide, ethidium diazide, and ethidium bromide, and to a lesser extent by the DNA-ethidium complex, the diazide-DNA or RNA adduct, and the monoazide-RNA adduct. DNA which had been photoaffinity labeled with either the propidium or the acridine moiety did not react. The antiserum recognition of DNA photoaffinity labeled with ethidium monoazide was based on the substituted phenanthridinium ring system of the parent ethidium, as evidenced by competition binding studies involving the free monoazido analog (EA1), the diazido analog (EA2), and the parent compound, ethidium bromide (EB). This approach and the sensitivity it provides should prove useful for identifying the distribution and fate of covalently bound drugs resulting from antiparasitic drug treatment, and for studying their roles in antiparasitic action.

Alkali lability and rapid initiation of excision repair following photoaffinity damage by ethidium azide.

DNA damage and repair provoked by ethidium azide (EA) photoaffinity labeling in mouse leukemia cells was studied by measuring sedimentation properties of nucleoids in neutral sucrose gradients, and it was found that the strand opening step was faster than that which followed damage of cells by ultraviolet (UV) light. The two insults were compared at levels of damage which gave the same overall rates of repair synthesis in intact cells and which required the same length of time to complete repair, as judged by the restoration of supercoiling of the isolated nucleoids. In the case of UV, single-strand breaks in DNA were detectable at 30 min, maximum at 2 h, and the superhelical properties restored at 21 h. With photoaffinity labeling, single-strand breaks were prominent immediately, even when photolabeling of cells was done on ice, but restoration of DNA supercoiling still required 21 h. Photolabeling of isolated nucleoids or isolated viral DNA with EA failed to introduce DNA strand breaks. However, it was discovered that photoaffinity labeling of DNA with EA resulted in alkali labile sites shown by single strand breaks produced on alkaline sucrose sedimentation or by alkali exposure followed by sedimentation on neutral formamide gradients. These results suggest that the drug attachment sites should be identifiable by the location of such single strand breaks.

DNA damage and repair in epithelial (mucous) cells and crypt cells from isolated colon.

Colon epithelium is made up of two general classes of cells, surface cells which are post-mitotic and crypt cells which contain the proliferative population. Their relative vulnerability to environmental damage and ability to perform DNA repair are important issues in colon carcinogenesis. DNA damage and repair was studied by the nucleoid sedimentation method in freshly isolated crypt cells for comparison with previous studies of post-mitotic surface epithelial cells. Suspensions of crypt cells were isolated from preparations of mouse colon by a series of sequential incubations in buffer containing 1.5 mM EDTA. Treatment of crypt cells for 30 min with 1.2 X 10(-6) M methyl methane sulfonate (MMS), photoaffinity labeling with 1 X 10(-6) M ethidium monoazide, lithocholic acid (2.5 X 10(-4) M) treatment for 1 h or X-irradiation at 1500 rads resulted in single-strand breaks in the DNA, which were repaired after 2 h of additional incubation. Interestingly, X-rays at 1000 rads and lithocholic acid (LA) (2.5 X 10(-6) M) after 30 min incubation failed to produce the detectable shift in nucleoid sedimentation characteristic of single-strand breaks, perhaps due to rapid repair by these proliferative cells. UV-irradiation failed to provoke strand incision as was also observed for the superficial post-mitotic cells in the previous studies. These studies showed the feasibility of studying DNA damage and repair processes in these two classes of colon epithelial cells in response to specific carcinogenic insult.

Petite and sectored induction in Saccharomyces cerevisiae by propidium iodide: synergistic effect of sodium dodecyl sulfate.

Sodium dodecyl sulfate (SDS) was examined for its effect on petite and sectored colony induction in Saccharomyces cerevisiae by propidium iodide (PI) and ethidium bromide (EB). 4-h cultivation with 100 microM PI and 100 micrograms/ml SDS resulted in virtually all plated cells growing as sectored colonies with no decrease in viability. Sectored colonies are mixed colonies comprised of respiratory deficient and competent cells believed to be derived from an unstable respiratory deficient cell. Further cultivation with PI and SDS prior to plating led to induction of complete petite colonies with a rapid decrease in viable cells. PI alone at this concentration exhibited weak induction of sectored colonies (maximum 12.3% at 8 h) and petite colonies (maximum 10.8% at 12 h), but SDS alone caused induction of neither. 50 microM PI had almost the same activity as 100 microM except for a delay in the induction of sectored colonies in the initial stage, and a decreased rate of petite colony induction. The effects of 20 microM PI and SDS were much lower than that by 50 microM and no inhibition of growth was observed. 10 microM PI was quite inactive even in the presence of SDS. Under resting conditions, 10 approximately 100 microM PI and 100 micrograms/ml SDS induced about 60% sectored colonies at 12 h incubation and more than 60% petite colonies at 24 h. After 6 h incubation, decrease in survival was also observed.

A protective effect of caffeine on the ethidium induced petite mutation in yeast.

A prerequisite for petite induction by ethidium bromide (EB) is an initial covalent attachment of the drug to cytoplasmic DNA. This DNA modification is thought to initiate repair processes. The repair inhibitor, caffeine, provided a protective effect against the ethidium induced petite mutation at caffeine concentrations known to inhibit the repair of UV damage in cytoplasmic DNA (Fig. 1). Mitochondrial DNA isolated from yeast exposed to EB in vivo was not as degraded in the presence of both drugs as with EB alone (Fig. 2).

Photolytic binding of the monoazido analog of ethidium to yeast mitochondrial DNA: competition by ethidium.

The [14C]-labeled monoazido analog of ethidium, 3-amino-8-azido-5-ethyl-6-phenylphenanthridinium chloride, when mixed with yeast cells and photolyzed, produced covalent adducts with both nuclear and mitochondrial DNA via the light-generated nitrene. The binding efficiency was about 12 times higher in mitochondrial than nuclear DNA. Moreover, the parent ethidium bromide at a 5-fold excess was an effective competitor for the binding of the monoazide analog with mitochondrial DNA, but not with nuclear DNA.

Antagonism by propidium of petite induction by ethidium and ethidium azide in Saccharomyces cerevisiae.

Propidium, a phenanthridinium dye similar to ethidium, did not induce petite mutations in non-growing yeast cells in contrast to ethidium. Combined exposure to ethidium and an excess of propidium for periods up to 2 h resulted in the expected petite induction expressed after subsequent plating on growth medium. As incubation was continued with propidium, the numbers of petites declined on subsequent plating whether the drug had been added before, during, or after the mutagenic treatment by ethidium. Propidium decreased petite induction by the monoazide analog of ethidium when applied before but not after photolytic attachment of the drug.

Induction of respiratory deficient mutants in Saccharomyces cerevisiae by mono- and diazido analogs of ethidium.

Mono- and diazido analogs of ethidium when photolyzed with yeast cells were highly effective in inducing respiratory deficient (RD) mutants. The monoazide was more mutagenic, though slightly less photosensitive, and under the concentrations and conditions used, both required photolysis to be significantly mutagenic. Ethidium bromide (EB) competed with either its mono- or diazide analog for RD induction when applied before, but not after, the photolysis step. This suggested that the initial mutagenic binding sites for azides were identical with those of EB. There was no self-rescue or recovery in azide mutagenesis in contrast to EB. Furthermore, recovery from azide mutagenesis could not be provoked by EB. This confirmed a simple competition between binding of EB and its azide analogs to account for the prevention by EB of the azide induced mutations.

Co-mutagenic effects of propidium on petite induction by ethidium in Saccharomyces cerevisiae.

Propidium, whose structure is closely related to ethidium bromide, induced a low level of petites in yeast, but only at high concentrations with long incubation time, and only in growth medium. When added to growing cells, propidium also caused a large increase in petite induction by ethidium even at submutagenic concentrations of ethidium. Incorporation of adenine into DNA was inhibited by propidium in mitochondria but not in nuclei. Propidium by itself had no effects on fragmentation of pre-existing DNA, but enhanced mitochondrial DNA degradation provoked by ethidium. The proportion of suppressive clones occurring among the petites from ethidium treatment was reduced by the presence of propidium. All of these results indicated that propidium treatment led to degradation of the mitochondrial DNA in petites induced by ethidium but not in native (intact) mitochondrial DNA, nor in spontaneous petite colonies. The results are discussed in terms of possible mechanisms of modulation of petite induction.