Hugo J. Niggli*^a,c, Corinne Scaletta^a, Yan Yu^b, Fritz-Albert Popp^b and Lee Ann Applegate^a

^aDepartment of Obstetrics, Laboratory of Oxidative Stress and Ageing, University Hospital, CHUV
CH-1011 Lausanne, Switzerland

^bInternational Institute of Biophysics, IIB, ehm. Raketenstation, Kapellener Strasse
D-41472 Neuss, Germany

^c*Address for correspondence : BioFoton AG, P.O. Box 28, CH-1731 Ependes, Switzerland
E-mail:biofoton@mail.swissonline.ch

Dedicated to the memory of Alexander Gurwitsch on the occasion of his 125th birthday.

At the beginning of this century, Alexander Gurwitsch, born 125 year ago in Russia, suggested that ultraweak photons transmit information in living systems [1] as summaraized recently [2]. Although the results of Gurwitsch were refuted by Hollander and Klaus [3] and the interest for this subject declined in the following decades, the presence of biological radiation was re-examined with the development of photomultiplier tubes in the mid-fifties by Facchini and co-workers [4]. In the sixties most of the work on ultraweak photon emission was performed by Russian scientists [5-7], while in Western countries several pioneers, Quickenden in Australia [8], Popp in Germany [9] and Inaba in Japan [10], independently developed methods for ultraweak photon measurements in a variety of different cells by the use of a extremely low noise, highly sensitive photon counting system which allows maximal exploitation of the potential capabilities of a photomultiplier tube. In the meantime it is commonly agreed that plant, animal and human cells emit ultraweak photons often called biophotons [11-17]. From these and additional investigations different origins for this very weak radiation have been proposed.

Most investigators think that very weak radiation results from radical reactions which can be produced by biological events such as lipid peroxidation. In studies of microsomal lipid peroxidation [18,19], it has been shown that the amount of malonaldehyde production and the intensity of emitted light are related to each other. Based on these studies, Inaba and co-workers proposed [20] that oxygen dependent light emission in rat liver nuclei was caused most likely by lipid peroxidation in the nuclear membrane. As discussed in detail by Cadenas and Sies [21], free radical decomposition of lipid hydroperoxides leads to the formation of excited chemiluminescent species by the self-reaction of secondary peroxy radicals formed from lipids, producing either singlet molecular oxygen or excited carbonyl groups.

However, there also exists a very interesting model published in 1983 by Nagl and Popp [22] suggesting that there is a negative feedback-loop in living cells which couples together states of a coherent ultra-weak photon or biophoton field and the conformational state of the cellular DNA. The authors assume photon transfer or non-radiation chemical pumping from the cytoplasmic metabolism which results in changes of the DNA conformation via exciplex/excimer formation. Their hypothesis is based on experimental data reviewed by Birks [23] who also suggested these excimers as precursors of the pyrimidine photodimers which play a key role in the radiation damage of DNA and has been most recently proposed as biomolecular signals in communication [24]. It was hypothesized that there exists an effective intracellular mechanism of photon absorption in normal human cells [25]. After UV-light irradiation of cells, pyrimidines absorb the energy in order to form pyrimidine dimers which could be responsible for influencing metabolic and cellular events as was recently shown for photodimer induced melanogenesis [26].

Tilbury discussed in the multi-author review of van Wijk [27] that ultraweak photon emission has been detected in both the visible and ultraviolet region. Radiation in the visible region appears to be due to excited carbonyl groups and/or excited singlet oxygen dimers arising from lipid peroxidation, which in turn are associated with an increase in various reactive oxygen species such as the superoxide anion, hydrogen peroxide, hydroxyl radical and singlet oxygen. There is also substantial evidence for DNA playing a key role in these emissions as discussed above [9,28,29]. This macromolecule may especially be involved in the emission of ultraweak photons by the cell in the UV-region of the spectrum [27].

Recently, experiments with cultured human cells were reported [25] in which normal and DNA excision repair deficient Xeorderma pigmentosum of complemation group A (XPA) cells were UV-irradiated in Dulbecco's modified Eagle's medium (DMEM) and Earle's balanced salt solution (EBSS) and assessed for ultraweak photon emission. There was some evidence of induced photon emission from normal cells in Earle's balanced salt solution, but clear evidence of a fluence-dependent emission in XPA cells in medium and in EBSS were seen. Overall, it was clear that an important difference between normal and XPA cells was present and it is possible that XPAn cells are less able to absorb ultraweak photons in contrast to normal cells.

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 [30]. They reported morphological and biochemical evidence for the fibroblast stem-cell differentiation system in vitro. They showed that normal human skin fibroblasts in culture spontaneously differentiate along the cell lineage of mitotic fibroblasts (MF) MFI-MFII-MFIII and post-mitotic fibroblasts (PMF) PMF IV-PMFV-PMVI-PMFVII. Additionally, they developped methods to shorten the transition period and to increase the frequency of distinct post-mitotic cell types using physical or chemical agents such as mytomycin C, 5-fluorouracil, and 5-bromo-2-deoxyuridine, that have been reported to induce differentiation in a variety of cell systems, as we have recently summarized [31-36]. Mitomycin C is an effective chemotherapeutic agent for several cancers in man; this effect is probably related to the interaction with DNA leading to DNA-DNA crosslinks and DNA-protein crosslinks. We have previously demonstrated that the mitomycin C-treatment of three different normal human fibroblast strains (CRL 1221, GM 38 and GM 1717), frequently used in mutation, transformation, and aging research, induces characteristic morphological changes in the fibroblasts and brings about specific shifts in the [35S]methionine polypeptide pattern of total cellular proteins [31]. These results support the notion that mitomycin C accelerates the differentiation pathway from mitotic to postmitotic fibroblasts. Using this system, we were also able to demonstrate that no significant difference exists in the rate and the extent of the excision-repair response to thymine-containing pyrimidine dimers following UV-irradiation shortly after mitomycin C treatment of distinct strains of human skin fibroblasts and in the mitomycin C-induced PMF stage of these cells [31]. In addition, aphidicolin inhibits excision repair of UV-induced pyrimidine photodimers in low serum cultures of mitotic and mitomycin C-induced postmitotic fibroblasts of human skin [32]. In view that fibroblasts play an essential role in skin aging and wound healing, our results imply that the fibroblast differentiation system is a very useful tool in order to elucidate the efficiency of growth factors.

Three different lots of bovine bone growth factors (BP2, 3 and BP5) in cryotubes (lyophilised; 1 mg/tube) were provided by Jeff Moehlenbruck (Sulzer Biologics, Inc., Austin, Texas, USA). In the following experiments factors BP2, BP3 and BP5 were tested at a concentration of 5 ng/ml. The mitotic cells were treated one week, while the postmitotic cells were incubated with these growth factors during two weeks. The growth factors were also added during treatment with mitomycin C to put the cells in post-mitotic status.

Before irradiation the cells were rinsed twice with 3 ml phosphate-buffered saline (PBS), covered with 4ml PBS and were irradiated as monolayers in tissue culture dishes at room temperature. For UVA radiation a UVASUN 3000 lamp (Mutzhas, Munich, Germany) was used at a dose rate of 300-600 Wm^-2 at a distance of 60-90 cm. Irradiation periods were on the average of 10-45 min maximum. The spectral output of the lamp was analyzed with a calibrated Optronic model 742 spectroradiometer (Optronics Laboratories Inc., Orlando, FL, USA). Before and after each experiment radiation fluences were monitored by an International Light Radiometer (IL 1700, calibrated against the spectroradiometer). The emission spectrum was between 340 and 450 nm with a maximum at 380+ 30 nm and the cells were then frozen in liquid nitrogen as described above until measurements on ultraweak photon emission.

For ultraweak photon emission measurements different fibroblasts samples were transported in liquid nitrogen. Some cells were transported fresh in T-75 flasks and used for ultraweak photon emission. The cells were thawed and were diluted in a volume of 10 ml Dulbecco's modified Eagle's medium, from which phenol red was omitted, and were transferred to a quartz sample glass (inside dimensions 2.2x2.2x3.8 cm3; wall thickness 0.15 cm). Detection and registration of spontaneous and light-induced emitted photons was accomplished at 25° C as described before [25]. The test samples were kept in a dark chamber in front of a single photon counting device equipped with an EMI 9558 QA photomultiplier tube (diameter of the cathode 48mm, cooled to -25° C). This high-sensitivity photon counting device is described in detail by Popp and co-workers [9,38] and measures photon intensities as low as 10-17 W in the range between 220 and 850 nm. Signal amplification is normally 106-107 and dark count ranges between 10-15 counts per second (cps). Maximum efficiency of the S20-cathode is 20-30% within the range of 200-350nm (maximum is at 250 nm), decaying almost linearly down to 0% at a wavelength of 870 nm and mean quantum efficiency in the entire spectral range is about 10%. The integral intensity values within each interval of 40 ms was stored and processed by an interfaced computer. For light induced emission experiments, irradiation of the test sample was performed perpendicular to the detecting direction with focused white light from a 75-W Xenon lamp. Each measuring cycle was started by irradiating the sample for 5s. The measurements for the monochromatic light induction was performed by changing the spectrum of the inducing light through a monochromomator (PTI, Hamburg, Germany) from 300 to 450 nm (25nm interval).

3. Results and Discussion

Proliferation of fibroblasts was determined by counting cultured cells following a one to two week treatment with 5 ng/ml DMEM of each lot of bone growth factor. The percentage of increase of cell proliferation was between 0 and 60% as shown in Table 1.

We have previously found that postmitotic fibroblasts are significantly reduced in UV-induced ornithine-decarboxylase responses [33]. In skin fibroblasts from a patient with Xeroderma pigmentosum (XPA) we detected a slight increase in the rate of thymine dimer induction formed after UV-irradiation of the MMC-induced postmitotic stage three weeks after MMC-treatment [31]. For ultraweak photon emission, our experiments with cultured human cells in which normal and XPA cells were UV- irradiated showed an important difference between normal and XP cells for the absorption of photons [25]. Since the white light induction for ultraweak photon emission in the differentiation system with XPA cells was very similiar in both cell lines (see Fig. 1), we decided to test the growth factors with the still commercially available skin Xeroderma Pigmentosum fibroblasts originating from the Human Genetic Mutant Cell Respository (XPA; GM05509A).

In previous experiments with normal cells we have found that white light-induced photon emission dynamics are identical after successive irradiations of approximately 1 min intervals, and even several cycles of illumination and measurement do not quantitatively change the emission intensity for several hours [28]. The light-induced emission curves are hyperbolic as we have reported most recently with mouse melanoma cells [29]. These results confirm several previous investigations in plant and mammalian cells [11,15,16]. As interpreted by Li and Popp [38,39 ], the hyperbolic decay kinetics found in living systems after pre-illumination with white light may indicate coherence of ultraweak photons due to collective excitation of nucleic acids within the DNA of the investigated fibroblasts.

In the first experiments with human XPA skin fibroblasts it was found that measurable amounts of ultraweak photons can be achieved with cells densities as low as 2-3x104 cells /ml (data not shown). At these cell densities, control values after irradiation with a white light source for induction of ultraweak photons were determined in mitotic and postmitotic fibroblasts from fresh cells and compared to those of frozen cells. The results are shown in Table 2.

Although there was a small increase in the induced photon emission from fresh cells, most probably due to the stress condition of the transport, it can be concluded that the frozen cells showed the same type of emission. Similiar results were obtained for monochromatic induction of the fibroblasts in the range of 300-450nm as shown for MMC- mitotic cells in Fig. 2.

In a previous report [25], we found a slight increase in the dark count rates in postmitotic fibroblasts in XPA cells in contrast to normal fibroblasts. Therefore, the dark count rate at a cell density of 3.6x104 cells /ml in untreated and growth factor treated postmitotic fibroblasts was determined (data not shown) and this increase of the dark count rate in postmitotic cells was confirmed. From these dark count rates a decrease percentage of the accelaration of MMC-induced differentiation could be measured and it can be concluded, that the growth factors reduce the accelaration of differentiation in post-mitotic cells induced by MMC by a factor of 10-30% (data not shown).

As shown in Fig. 2 the most significant increase in ultraweak photon emission after monochromatic irradiation of fibroblasts between 300-450 nm was found at 350 nm. Therefore, the induced photon emssion after 350 nm light induction at a cell density of 3.6x104 cells /ml in untreated and growth factor treated MMC-mitotic fibroblasts was determined. In addition, the cells were treated with 400 kJ/m2 UVA as described elsewhere [37]. For mitotic cells, the results are shown in Table 3 showing an improvement of photon absorption ability in XPA-cells for the growth factors in the efficiency range of BP5>BP3>BP2.

For MMC-treated mitotic fibroblasts similiar results have been obtained as shown in Table 4 as well as for MMC induced-postmitotic fibroblasts (data not shown).

We have previously found that after white light induction there is a small decrease from mitotic to postmitotic fibroblasts from XPA-cells while there is in MMC-treated normal fibroblast a significant increase [28]. Additionally we detected that normal cells absorb UV-photons more efficiently tha XPA-cells [25]. As shown in Table 4, growth factor treatment indeed increase the monochromatic induction in these cells similar to normal cells. Most interestingly, UVA irradiation in mitotic and postmitotic cells significantly reduce the monochromatic induction of ultraweak photons. It can be assumed, therefore, that the decrease in mitotic untreated and MMC treated cells which has been exposed to growth factors is due to absorption of photons, emphasizing a return to normal basal status.

As finally summarized, our results indicate that the tested growth factor solutions have an effect on growth and ultraweak photon emission in the differentiation model of human skin fibroblasts and provides a new and powerful non-invasive tool for the development of skin science. Its high sensitivity can be applied in all fields of skin research, in investigating skin abnormalities and in testing the effect and efficacy of regenerative, anti-aging and UV-light protective agents at very low concentrations.

We are indebted to Jeff Moehlenbruck (Sulzer Biologics, Inc., Austin, Texas, USA) for providing us with growth factors. This study was funded in part by grants from the Swiss League Against Cancer (KFS 695-7-1998) and the Erwin Braun Foundation. HN was given generous support by the H +M Kunz foundation (Urdorf, Switzerland) for which this study could be accomplished. We are also indebted to Dr. Max Bracher (Cosmital SA, Research Company of Wella AG, Darmstadt, Germany) for technical support.