Human peripheral mononuclear cell responses to UV damage are affected by radiation-induced changes in plasma.

To evaluate the effects of environmental or therapeutic stress adequately, it is important to study cells or tissues under conditions that simulate as closely as possible the in vivo environment. To determine whether the responses of irradiated cells are significantly affected by radiation-induced changes in plasma, human mononuclear cells were isolated from peripheral blood and cultured in their autologous plasma. Freshly isolated cells were irradiated in phosphate-buffered saline. The plasma was irradiated separately. Irradiation of the plasma suppressed mitogen-induced DNA synthesis in unirradiated cells. For cells that were UV-damaged and subsequently stimulated with mitogen, DNA synthesis was enhanced by irradiation of the plasma. Medium in which irradiated cells had previously been incubated enhanced DNA, synthesis in unirradiated cells that had been mitogen stimulated but did not affect the UV-induced shutoff of DNA synthesis in replicating cells or unscheduled DNA synthesis in irradiated cells.

The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis.

Ethidium Bromide (EtBr) is sometimes added to running buffer during the separation of DNA fragments by agarose gel electrophoresis. It is used because upon binding of the molecule to the DNA and illumination with a UV light source, the DNA banding pattern can be visualized. The mode of binding of EtBr is intercalation between the base pairs. This binding changes the charge, weight, conformation, and flexibility of the DNA molecule. Since DNA molecules are sized by their relative movement through a gel compared to a molecular weight standard, mobility measurements can be critical to size determinations. After running two identical gels, one without EtBr and one with 0.25, 0.5, 0.75 or 1.0 microgram/mL EtBr in the running buffer, the mobilities of lambda HindIII DNA fragments were compared. The mobility of DNA was always less in the gels with EtBr. Using the reptation theory equation, which describes the mobility of DNA molecules through gel, changes in frictional coefficients were calculated. It was determined that the change in frictional coefficients brought about by the addition of EtBr is directly proportional to the fraction of base pairs of a fragment bound to EtBr. This change in friction is greatest in the largest fragments, which suggests that the stiffening of the molecule by the EtBr binding is the cause for the decreased mobility.

Suppressed DNA repair capacity of peripheral lymphocytes in pregnant women.

The ability to repair potentially carcinogenic lesions was measured for peripheral lymphocytes isolated from women in the first, second and third trimesters of pregnancy and 6 weeks post-partum. Freshly isolated lymphocytes were damaged with 254 nm ultraviolet radiation and allowed to perform repair in their autologous plasma. DNA repair capacity during pregnancy was over 50% lower than that measured 6 weeks post-partum. No significant difference in repair capacity was observed among the three trimesters. Mitogen-stimulated DNA synthesis was also measured for cells cultured in their autologous plasma. There was no significant difference in thymidine incorporation between cells collected during pregnancy (1.02 +/- 0.17 cpm/10(6) cells) and cells collected 6 weeks post-partum (1.45 +/- 0.94 cpm/10(6) cells). The results are, however, consistent with alterations in the immune response during pregnancy.

An assay for monitoring response to therapy in cancer patients.

Immunosuppression is characteristic of patients with advanced malignant neoplasms. However, none of the simple assays for immunocompetence have been found to provide results which correlate with the patients' responses to therapy. The data reported here indicate that this may be because there are artifacts in data from earlier studies which have obscured such a correlation. A simple assay which removes previously unrecognized sources of error is shown to generate data which correlate well with patient responses. In this assay, lymphocytes collected from patients with malignant solid tumors were stimulated with mitogen in their autologous plasma. The amount of radioactive thymidine incorporated during replicative deoxyribonucleic acid (DNA) synthesis was corrected to remove sources of error ignored in the previous studies. A significant improvement in mitogen-stimulated synthesis was observed within two months for patients entering remission. For patients not responding to therapy, there was a progressive deterioration in mitogen-responsiveness.

Correlates of plasma cortisol and DNA repair in human peripheral lymphocytes: suppression of repair in women taking estrogen.

We have examined the relationship between total plasma cortisol concentration and DNA repair capacity in human peripheral lymphocytes cultured in vitro; the data indicate that high concentrations of cortisol (> 20 micrograms/dl) inhibit DNA repair. The inhibitory effect can be abrogated by the addition of RU38486, a cortisol antagonist. In addition, we compared plasma cortisol concentration and in vitro DNA repair capacity in 52 healthy individuals. Females on therapeutic estrogen (oral contraceptive or estrogen replacement therapy) had significantly elevated plasma cortisol and suppression of DNA repair capacity.

Tanning salon exposure suppression of DNA repair capacity and mitogen-induced DNA synthesis.

Mitogen responsiveness and the capacity to repair genetic damage were measured in lymphocytes collected from young, healthy, adult Caucasians immediately before exposure in commercial tanning salons and again 24 h after exposure. For every individual studied, tanning exposure produced significant inhibition of phytohemagglutinin-induced mitogenesis or of the ability to repair DNA lesions by unscheduled DNA synthesis. The results imply that such exposure could: (1) pose a significant hazard for individuals who are already immunosuppressed (e.g. cancer patients, AIDS patients or carriers of latent HIV) and (2) increase the carcinogenic effects of environmental mutagens.

Electrophoretic separation of nucleic acids: evaluation by video and photographic densitometry.

The separation of DNA by gel electrophoresis provides a rapid method for determining size distributions of DNA in solution. Densitometric scanning of photographs of gels has been the standard method of analysis of agarose gels. However, analysis of photographs is complicated by the non-linear response of photographic film. Charged-coupled device video cameras have become popular for quantitative densitometry and we have used a charge-coupled device camera to image agarose gels to quantitate DNA damage. We compare video and photographic densitometry for quantitation of ultraviolet radiation (UV)-induced DNA damage and find that the two methods give equivalent results.

Quantitative fluorescence of DNA-intercalated ethidium bromide on agarose gels.

Techniques for analyzing DNA distributions on agarose gels are examined by both two-dimensional and one-dimensional methods. It is demonstrated that very large errors in DNA concentration occur in such analyses unless (i) the electrophoresis is performed in a careful, reproducible manner, (ii) the films are calibrated with an internal standard, (iii) high resolution densitometry is used for analyzing the films, and (iv) appropriate background controls are used to determine the baselines for integration. Two-dimensional scanning produces more accurate results than one-dimensional scanning, but in cases where the bands are relatively uniform, the one-dimensional analysis gives good results. A technique for determining accurate distributions is described.

Quantitative assay for evaluating immunocompetence and DNA repair capacity.

Incorporation of radioactive thymidine into newly synthesized DNA is the basis of an assay frequently used to study immunosuppression in cancer patients. It has also been used to measure the amount of excision repair performed by non-replicating cells damaged by carcinogens. For human lymphocytes (and probably other cell types), these assays are unreliable as they are currently being performed. We report a modified assay that allows accurate comparisons of the immunocompetence and DNA repair capacity of different individuals. With this assay, cells can be studied in their autologous plasma and the role of biological response modifiers can be assessed.

Effect of ultraviolet radiation on the Bacillus subtilis phages SPO2, SPP1 and phi 29 and their DNAs.

A comparative study of the effects of ultraviolet radiation on three Bacillus subtilis phages is presented. Phages phi 29, SPP1 and SPO2c12 or their DNAs were irradiated by UVC (254 nm) and quantum yields for inactivation were calculated. For each phage, the purified DNA was found to be more sensitive than the intact virus when assayed in a uvr+ host. The data imply that this is because transfecting DNA is repaired less efficiently than DNA of the intact phage; rather than because of differences in sensitivity to lesion production. Even though phi 29 has the smallest target size of the three phages, phi 29 and its DNA are the most sensitive. Phages SPO2 and SPP1 code for gene products which complement the repair system of the host. The transfecting DNA of phage SPP1 is extremely sensitive to UV damage when assayed in a uvr-host. This is attributed to the fact that in transfection SPP1 DNA must undergo recombination for productive infection to occur. The recombination process strongly interferes with the repair of damaged DNA.

Serum effects on DNA repair in human cells.

Freshly isolated human lymphocytes were used to determine how serum supplements affect cellular capacity to repair UV damage. Repair capacity was always found to be greatest in medium supplemented with autologous plasma. Variability in repair capacity among individuals was greater in serum supplemented medium than in unsupplemented medium. Thus, in vitro cellular responses will most accurately represent in vivo responses if autologous serum factors are present in the culture medium. This is of particular importance in studies attempting to correlate DNA repair capacity with age or susceptibility to carcinogenesis.

Sensitivity of carcinogen-treated DNA to inactivation by ultraviolet radiation.

The DNA of bacteriophage SPO2c12 was treated with methylmethane sulfonate (MMS), beta-propiolactone (BPL), 2-anthramine (AA) or benzo[a]pyrene (BP) and then exposed to 254-nm radiation. Competent Bacillus subtilis host cells were transfected with DNA subjected to the carcinogen-UV treatment or with DNA treated with carcinogen only. Survival curves were obtained for loss of plaque-forming ability as a function of UV dose. The UV sensitivity of DNA treated with MMS, BPL or AA was not significantly different from that of untreated DNA. The results indicate that in competent B. subtilis the pathways for repair of alkylating agent damage and for repair of UV damage are probably different.

Inhibition of unscheduled DNA synthesis in human lymphocytes by chemical carcinogens.

Freshly isolated human peripheral lymphocytes were treated with an alkylating agent immediately after collection and subsequently treated with UV radiation. This system was used because it represents a method for assaying damage in cells immediately after their removal from the host. The amount of UV-induced repair was measured as unscheduled DNA synthesis (UDS) by incorporation of [3H]deoxythymidine into the cellular DNA. The alkylating agents beta-propiolactone (BPL) and methyl methane-sulfonate (MMS) inhibited UDS at concentrations of 0.08 mM and 0.6 mM, respectively. Lower concentrations had no effect. Lymphocytes allowed to remain in culture medium after treatment with the alkylating agents did not recover the ability to perform UV-induced UDS even when cells were irradiated 48 h after carcinogen treatment. The decrease in UV-induced UDS resulting from alkylating agent treatment could not be attributed to cell death.