Ultraweak photon emission in assessing bone growth factor efficiency using fibroblastic differentiation.

Photons participate in many atomic and molecular interactions and changes. Recent biophysical research has shown the induction of ultraweak photons in biological tissue. It is now established that plants, animal and human cells emit a very weak radiation which can be readily detected with an appropriate photomultiplier system. Although the emission is extremely low in mammalian cells, it can be efficiently induced by ultraviolet light. In our studies, we used the differentiation system of human skin fibroblasts from a patient with Xeroderma Pigmentosum of complementation group A in order to test the growth stimulation efficiency of various bone growth factors at concentrations as low as 5 ng/ml of cell culture medium. In additional experiments, the cells were irradiated with a moderate fluence of ultraviolet A. The different batches of growth factors showed various proliferation of skin fibroblasts in culture which could be correlated with the ultraweak photon emission. The growth factors reduced the acceleration of the fibroblast differentiation induced by mitomycin C by a factor of 10-30%. In view that fibroblasts play an essential role in skin aging and wound healing, the fibroblast differentiation system is a very useful tool in order to elucidate the efficacy of growth factors.

Glutathione response after UVA irradiation in mitotic and postmitotic human skin fibroblasts and keratinocytes.

Since Hayflick's pioneering work in the early sixties, human diploid fibroblasts have become a widely accepted in vitro model system. Recently, Bayreuther and co-workers extended this experimental approach showing that fibroblasts in culture resemble, in their design, the hemopoietic stem-cell differentiation system. They found that the chemical agent mitomycin C accelerates the differentiation pathway from mitotic to postmitotic fibroblasts. We measured the response of endogenous glutathione levels after UVA irradiation (320-400 nm) in mitotic and mitomycin C-induced postmitotic human skin fibroblasts and foreskin-derived keratinocytes. The initial levels in mitotic foreskin derived human fibroblasts were 14.4 nmol glutathione per mg protein, whereas a 30% higher value was obtained in matching foreskin-derived keratinocytes. Similar elevated levels of this important intracellular free radical scavenging system were found in fibroblasts of a donor suffering from xeroderma pigmentosum. Furthermore, three to four times higher levels of glutathione in mitomycin C-treated mitotic fibroblasts have been determined. In mitotic skin fibroblasts, UVA irradiation resulted in a depletion of glutathione up to 90% following a fluence of 1.0 MJ/m2 UVA radiation. Higher initial glutathione levels were found in keratinocytes and mitomycin C-treated skin fibroblasts. In these fibroblasts a 70% depletion was detected and a much lower depletion (10-20%) was seen in some keratinocyte cell lines following fluences up to 1.0 MJ/m2. The depletion in skin fibroblasts was retained after 24 h following a fluence of 0.75 MJ/m2 UVA light. In view of the fact that glutathione has been shown to be involved in a variety of metabolic processes and plays a role in cellular protection against UVA radiation, our results imply that the fibroblast differentiation system is a very useful tool to unravel the complex mechanism of UVA-induced oxidative stress.

Aphidicolin inhibits excision repair of UV-induced pyrimidine photodimers in low serum cultures of mitotic and mitomycin C-induced postmitotic human skin fibroblasts.

The rates of formation and excision of UVC light-induced cyclobutane-type pyrimidine photodimers were determined in cultures of foreskin-derived normal human fibroblasts in mitotic (MF) and mitomycin-C (MMC)-induced postmitotic fibroblasts (PMF). Characteristic morphological changes support the notion that MMC accelerates the differentiation pathway from MF to PMF. In cultures treated with aphidicolin, I am able to show that this inhibitor of alpha and/or delta polymerases significantly inhibits the repair of pyrimidine photodimers in foreskin-derived mitotic and MMC-induced postmitotic fibroblasts in low serum cultures (0.5%) following UVC irradiation. Over the concentration range of 0-2 micrograms/ml of aphidicolin, there is a strong concentration-dependent inhibition of repair in cells treated with 10 J/m2 of UVC and incubated with aphidicolin during the post-incubation time (0-24 h). The results demonstrate that pyrimidine photodimers are repaired in low serum cultures by an alpha- and/or delta-polymerase-dependent pathway. These data also imply that the fibroblast differentiation system is a very useful tool to unravel the complex mechanisms of UV-induced DNA damage and repair.

Artificial sunlight irradiation induces ultraweak photon emission in human skin fibroblasts.

Photons participate in many atomic and molecular interactions and changes in the physical universe. In recent years sophisticated detection procedures for the measurement of ultraweak photons in a variety of different cells have been performed leading to the conclusion that plant, animal and human cells emit ultraweak photons. Using an extremely low-noise, high-sensitive photon-counting system, which allows maximal exploitation of the potential capabilities of a photomultiplier tube, ultraweak photons were quantitated in human skin fibroblasts. It was found that light from an artificial sunlight source induces ultraweak photon emission in these cells. However, the results demonstrate that this induction is significantly lower in normal fibroblasts compared with those obtained from a donor suffering from xeroderma pigmentosum disease group A, a disease characterized by deficient repair of DNA. The largest increase in ultraweak photon emission after UV exposure was measured in mitomycin-C-induced post-mitotic xeroderma pigmentosum cells which showed 10-20 times higher ultraweak photon intensities than mitotic UV-irradiated normal cells. These data suggest that xeroderma pigmentosum cells tend to lose the capacity of efficient storage of ultraweak photons, indicating the existence of an efficient intracellular photon trapping system within human cells.

Determination of cytosine-cytosine photodimers in the DNA of Cloudman S91 melanoma cells using high pressure liquid chromatography.

I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (less than 290 nm) and UV-B irradiation (290-320 nm) in the 2'-deoxy-[3H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol. 52, 519-524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10-20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.

Comparative studies on the correlation between pyrimidine dimer formation and tyrosinase activity in cloudman S91 melanoma cells after ultraviolet-irradiation.

We compared the induction of pyrimidine dimer densities after UV-irradiation in mouse melanoma cells before and after treatment with cholera toxin. Treatment with cholera toxin stimulated tyrosinase activity up to 50-fold, leading to a marked, visually apparent increase in cellular melanin concentrations. Irradiation of treated and untreated cells was therefore designed to establish whether intracellular melanin protected cells from UV-induced DNA damage. In experiments described here, we determined cytosine-thymine (C-T) as well as thymine-thymine dimer levels (T-T) by high pressure liquid chromatography in cholera toxin-treated and untreated Cloudman S91 mouse melanoma cells after irradiation with UVC (less than 290 nm) and UVB light (290-320 nm). Surprisingly, induction of melanization had no effect on the formation of pyrimidine dimers by UVC or UVB irradiation. These results indicate that de novo melanin pigmentation induced via the c-AMP pathway is not involved in protection against UV-induced thymine-containing pyrimidine dimers. In separate experiments, irradiation of toxin-treated and untreated mouse melanoma cells with UVC or UVB light produced a 20-30% lower dimer density compared to irradiated human skin fibroblasts. This finding suggests that melanin has some protection properties against UV-induced pyrimidine dimers, although the exact defense mechanism seems highly complex.

Mitomycin C-induced postmitotic fibroblasts retain the capacity to repair pyrimidine photodimers formed after UV-irradiation.

The formation and excision of UV-C light-induced cyclobutane-type pyrimidine photodimers were determined in cultures of human skin fibroblasts at time zero and several weeks following treatment with mitomycin C (MMC). Characteristic morphological changes of the fibroblasts and specific shifts in the [35S]methionine polypeptide pattern of total cellular proteins support the notion that MMC accelerates the differentiation pathway from mitotic (MF) to post-mitotic fibroblasts (PMF). No discernible difference could be detected between the fluence-response curves of pyrimidine dimers for untreated and MMC-treated repair-deficient xeroderma pigmentosum cells of group A. Furthermore we investigated the removal of pyrimidine dimers in 3 normal human skin fibroblast strains frequently used in mutation, transformation and aging research. We were able to demonstrate that no significant difference exists in the rate and extent of the excision-repair response to thymine-containing pyrimidine dimers following UV-irradiation shortly after MMC treatment of fibroblasts and in the MMC-induced PMF stage of these cells.

Cyclobutane-type pyrimidine photodimer formation and induction of ornithine decarboxylase in human skin fibroblasts after UV irradiation.

Cyclobutane-type pyrimidine photodimers as well as the induction of ornithine decarboxylase (ODC) may serve as biochemical markers of the mutagenic and carcinogenic effects of ultraviolet light (UV). For this reason, it is important to compare the formation of pyrimidine dimers with the induction of ODC in human skin fibroblasts after irradiation with UVC (200-290 nm) and UVB (290-320 nm). In our studies we determined cytosine-thymine (C-T) as well as thymine-thymine dimer yields (T-T) by high-pressure liquid chromatography in cultures of neonatal normal human foreskin-derived fibroblasts after irradiation with UVC and UVB light. It was found that the yield of dimerization and the ratio of T-T/C-T decreased from the UVC to the UVB region. Time-course studies of ODC-induction in the same cells indicated that the maximal activity after UVB irradiation was retarded compared to UVC exposure. For the UV-induced ODC-levels, however, no significant difference in maximal induction could be measured after UVC and UVB irradiation at fluences where comparable yields of thymine dimerization are produced. Similar ODC-maxima were obtained with strains from children, while cells from adults showed significantly less pronounced ODC induction, indicating that ODC-response decreases with age and may therefore be used as a marker of aging.

Cyclobutane-type pyrimidine photodimer formation and excision in human skin fibroblasts after irradiation with 313-nm ultraviolet light.

The formation and excision of 313-nm light-induced cyclobutane-type pyrimidine photodimers were determined in confluent cultures of human fibroblasts. A new method was developed for the resolution and determination of cytosine-thymine (CT) and thymine-thymine dimers (TT) by using sodium borohydride reduction and high-pressure liquid chromatography. This assay can detect as little as 1.8 TT or 5.6 CT per 10(8) daltons, levels induced in monolayers of human skin fibroblasts by doses of 1 and 2 kJ m-2 of 313-nm light, respectively. CT formation was 20% more efficient than TT formation in the physiological dose range of 2.25-15 k m-2 at 37 degrees C. Normal fibroblasts removed 61% TT within the first 8 h of incubation following a dose of 5.5 kJ m-2. CT was removed approximately twice as efficiently as TT during the same time period following exposure to 10 kJ m-2. The lack of removal of CT as well as TT observed in xeroderma pigmentosum fibroblasts indicates that the repair deficiency in these cells affects the repair of both classes of dimers.