Changes in cytokine content in the peripheral blood of volunteers after their exposure to polychromatic visible and infrared light

Anti-inflammatory, immunomodulating and wound-healing effects of visible and infrared (IR) radiation from laser and non-laser sources are widely used in current medicine. However, the role of pro- and anti-inflammatory cytokines in development of these effects has been poorly studied. A randomized, placebo-controlled, double blind study was made. Using ELISA, the content of 10 cytokines was studied in the peripheral blood of volunteers after a single and four daily irradiations of the sacral area (D = 15 cm) with polychromatic visible + IR polarized light (480-3400 nm, 12 J/cm2). The phototherapeutic sessions were accompanied by four blood exfusions for the study (to a total volume of 80 ml). In the control (placebo) group, irradiation was imitated, and blood samples of the same volume were drawn at the same time intervals as in volunteers of the main group. A fast decrease in the level of pro-inflammatory cytokines was revealed as soon as in 0.5 h after the irradiation. This level was retained until the end of the phototherapeutic course. At the parameters exceeding the norm, the contents of TNF-alpha, IL-6 and IFN-gamma fell, on average, by 34, 12 and 1.5 times, respectively. By the end of the course, the levels of IFN-gamma and of IL-12 decreased by 5 and 15 times, respectively. A fast decrease (by two-fold) was also characteristic of normal values of IL-6. Neither IL-1beta, nor IL-2 were detected in blood plasma of the examined people both before and after the irradiation. In parallel with a decrease in the proinflammatory factor levels the amount of anti-inflammatory cytokines was found to rise: that of IL-10--by 2.7-3.5 times in 0.5 h and at later terms at the initially normal parameters, and that of TGF-beta1--by 1.4-1.5 times at the initially decreased level. The IL-4 content did not change. A characteristic feature of the light effect was a fast rise of IFN-gamma amount--by 3.3-4.0 times in individuals with its initially normal level, with no changes in IFN-alpha content. The above-reported regularities of the light effects were also recorded at a direct (in vitro) irradiation of the examined volunteers' blood, as well as on addition of irradiated blood to a 10-fold volume of non-irradiated autologous blood, i.e. at a modeling of mixing, of a small amount of transcutaneously photomodified blood with its main circulating volume in the vascular bed of an irradiated person. Such a similarity of effects in blood following its irradiation in vivo and in vitro enables us to associate the fast changes of the cytokine content in the entire volume of peripheral blood with the transcutaneous photomodification of its small amounts, and with a "transfer" of the light effects by photomodified blood to the whole pool of circulating blood.     

Enhancement of fibroblast growth promoting activity of human blood after its irradiation in vivo (transcutaneously) and in vitro with visible and infrared polarized light

Visible and infrared (IR) irradiation of laser and non-laser sources has a pronounced wound-healing effect promoting tissue repair without hyperproduction of connective tissue elements. This effect develops as a consequence of local and systemic light effects, but many aspects of their mechanism have been yet unclear. In the present work, we have shown that in 0.5 h after irradiation of a small area of the volunteers' body surface with polychromatic visible + IR light (400-3400 nm, 95% polarization, 12 J/cm2) the amounts of PDGF and TGF-beta 1 in the blood serum increase, on average, by 20 and 43%, respectively. This effect is preserved for at least 24 h to be recorded only in volunteers with the initially normal and decreased levels of the growth factors; the initially elevated content of PDGF-AB decreases. Addition of such a plasma (2.5%) to the nutrient medium of primary cultures of human embryonal fibroblasts stimulates cell proliferation, on average, by 10 and 17%, but only in the case if the initial growth-promoting (GP) blood activity was low. Similar changes occur in parallel experiments following irradiation of blood samples of the same volunteers in vitro, as well as at mixing irradiated and non-irradiated autologous blood at the ratio 1:10 (v/v), i.e. at modeling a situation in the vascular bed, when the transcutaneously photomodified blood contacts with the rest of its volume. Similar changes in the blood GP activity under conditions in vitro were recorded as well after 4-9 daily phototherapy sessions. This allows us to suggest that changes in GP activity of circulating blood of the irradiated volunteers may be, to a large extent, the consequence of effect exerted on the blood by small amounts of transcutaneously photomodified blood. The obtained results are discussed in terms of light effect on wound healing and scar tissue formation, with regard to the authors' previous data on much higher GP of the irradiated blood in respect to keratinocytes, the fast decrease in proinflammatory cytokine levels, and the increase in IFN-gamma content.     

Changes in the expression of membrane markers and in the number of human blood monocytes after single and repeated courses of visible and infrared light at therapeutic doses

An attempt has been made to prove that the immunomodulating effect of therapeutic doses of polychromatic visible + infrared polarized (VIP) light at its application to a small body surface area is connected with a transcutaneous photomodification of a small amount of blood in superficial skin microvessels. For this purpose, in parallel experiments, using monoclonal antibodies, the membrane phenotype of circulating blood mononuclears was studied after irradiation of volunteers, of samples of their blood in vitvo, and of a mixture of the irradiated and non-irradiated autologous blood in a 1:10 volume ratio, thereby modeling events in vivo, when a small amount of the transcutaneously photomodified blood in the vascular bed contacts its main circulating volume. In this variant of experiment, a great similarity has been established of changes in expression of mononuclear membrane markers (CD3, CD4, CD8, CD20, CD16, HLA-DR and to a lesser degree of CD25); the ability has been proven of the photomodified blood to "translate" the light-induced changes to a much higher volume of non-irradiated blood, which might represent a mechanism of the systemic immunomodulating effect of phototherapy. Under conditions in vivo and in vitro, the most "reactive" were HLA-DR+, CD20+, CD16+, CD4+, and 0-cells. An increase of the total number of lymphocytes and monocytes has been shown by the end of the 10-day-long phototherapeutic course. The regulatory character of the single and course sessions of the VIP light on the blood immunocompetent cells is substantiated: depending on the initial state of the immune system, the VIP light can produce both stimulating and inhibitory effect on lymphoid cell subpopulations, which opens large possibilities of using this method for correction of immunological disturbances in diseases of different etiopathogenesis.     

Proliferation and tumorigenity of murine hepatoma cells irradiated with polychromatic visible and infrared light

In experiments in vitro, the effects of polychromatic visible (VIS) light combined with polychromatic infrared light (VIS-IR, 480–3400 nm) and the effects of the entire spectrum of VIS radiation (385–750 nm) on viability and proliferative activity of the murine hepatoma cells MH22a were studied. In experiments in vivo, changes in the tumorigenic properties of cells MH22a were studied after the same kinds of light exposure. It was shown that irradiation of hepatoma cells with two kinds of polychromatic light at a wide range of doses (4.8–38.4 J/cm²) did not lead to an increase in the number of dead cells for 24–72 h of cultivation and did not cause deceleration of the hepatoma cell proliferation; moreover, the VIS-IR light at a dose of 4.8 J/cm² and the VIS light at a dose 38.4 J/cm² even promoted more intense cell proliferation after 24 h. In cells irradiated with VIS-IR and VIS light, the proliferation index rose by 1.6 and 1.4 times, respectively, and the time of the cells’ number doubling decreased as compared with control. Studying the tumorigenic properties of irradiated tumor cells has shown that, for 30 days after transplantation to syngenic mice C3HA of hepatoma cells 24 h after their irradiation with VIS-IR light at a dose of 4.8 J/cm², the tumor volume decreased significantly (2.6–4.1 times) at all periods of observation, while the incidence of tumor formation decreased, whereas the survival of the tumor-bearing mice did not change. Transplantation of cells irradiated with the same light at a dose of 9.6 J/cm² did not lead to significant changes in the tumor volume, the tumor formation incidence, and animal survival. The main contribution to the antitumor effect of VIS-IR light seems to be made by the VIS component, as transplantation into mice of cells irradiated with VIS light alone at a dose of 38.4 J/cm² also stimulating proliferation of hepatoma cells in vitro resulted in a decrease of their tumorigenic properties. However, the IR component in the combined VIS-IR radiation enhanced the antitumor effect of the VIS light; as a result, it was manifested after use of doses eight times lower (4.8 J/cm²) than in the case of VIS light alone (38.4 J/cm²). Mechanisms of the decrease of tumorigenic properties of hepatoma cells after irradiation with polychromatic light at doses stimulating their proliferation in vitro are studied.     

Effect of the balanced inhibition of DNA and protein synthesis on the in vitro and in vivo proliferative activity of the lymphocytes]

Lymphocytes isolated from the rabbit peripheral blood were irradiated in vitro with 200, 400 and 1000 r doses and cultivated with phytohemagglutinin (PHA) doses and cultivated with phytohemagglutinin (PHA) at 37 degrees C during 48 hours. In several experiments cycloheximide, inhibiting protein synthesis, was added to the cells 60 minutes before and 30 minutes after irradiation. There was no apparent difference in the viability of irradiated cells with or without cycloheximide. The ability of lymphocytes of the popliteal lymph nodes for proliferation after PHA injection into one of the hind foot-pads of the irradiated mice was studied, as well. The injection of cycloheximide or puromycin into one of the hind foot-pads immediately after irradiation of the animals augmented the proliferation of lymphocytes in this extremity in comparison with contralateral one, 1.5-2 times. Cytosine arabinoside, inhibiting DNA synthesis, was not effective under these conditions.     

The effect of UV irradiation and of UV-irradiated autologous blood on the functional state of human peripheral blood lymphocytes

The effect of UV irradiation (UVI, 254 nm) and of UV-irradiated autologous blood on the spontaneous and mitogen-induced DNA-synthetic activity of intact lymphocytes has been studied. Lymphocytes were isolated from nonirradiated and irradiated blood, and from the mixture of UV-irradiated blood with the intact one in the volume ratio close to that in the blood stream during UV-irradiated blood autotransfusion (1:10, 1:40, 1:160). It has been shown that UVI of the whole blood caused in some donors the increase in spontaneous DNA synthesis, while in others the decrease or no statistically significant changes were observed. The analysis of the results obtained shows an inverse relation of the UVI effect to the initial level of spontaneous DNA synthesis (r = -0.68). In contrast to direct UVI effect, an addition of UV-irradiated blood to the autologous intact one resulted in an increase in spontaneous DNA synthesis in lymphocytes of all the samples examined. A 7-day cocultivation of lymphocytes, isolated from irradiated and nonirradiated blood samples, revealed a 1.8 times increase compared to the calculated value. The mitogen-induced DNA synthesis has a low sensitivity to UV rays, since the mitogens and the irradiation of optical range have presumably the common targets. It is assumed that photomodification of HLA-D/DR antigens can be a trigger mechanism for activation of immunocompetent cells by UVI.     

Extreme ultraviolet radiation-sensitivity of respiratory adaptation in Saccharomyces cerevisiae cells during transition.

The respiratory adaptation process in both wild-type and UV-sensitive strains of $\text{Saccharomyces}\text{cerevisiae}$ was sensitive to small doses of UV-radiation (10 and 0.7 J/m², respectively). These doses of irradiation were ineffective in arresting induced synthesis of acid phosphatase and catalase. Exposure of the irradiated cells to visible light (370 – 800 nm) could completely restitute the impaired respiratory adaptation process. UV irradiation at these doses affected DNA and RNA synthesis in maturing mitochondria in both the yeast strains. The UV-induced block could however be eliminated by exposure of the cells to visible light. These results suggest that the lesion in the UV-induced block in the respiratory adaptation may be in the DNA of promitochondria.     

Light-induced denaturation of bacteriorhodopsin solubilized by octyl-beta-glucoside.

The structural stability of bacteriorhodopsin (bR) solubilized by octyl-beta-glucoside was studied by measuring the denaturation kinetics under visible light irradiation and in the dark. The denaturation of bR solubilized by 50 mM octyl-beta-glucoside was very slow at room temperature when it was left in the dark. However, its spontaneous denaturation was accelerated when the solubilized bR was irradiated by visible light. The denaturation kinetics under visible light irradiation and in the dark could be well described by a single decay constant. The activation energy for the denaturation of bR was estimated from the temperature dependence of decay time constants. The activation energy under visible light irradiation was 12.5 kcal/mol, which was much smaller than the corresponding value in the dark, 26.2 kcal/mol. These results strongly suggest that some of the photointermediate states are less stable than the ground state of bR. The critical temperature and the activation energy for denaturation of bR in the solubilized state were much lower than those in the 2D crystalline state. Comparing the denaturation behavior in the 2D crystalline state and that in the octyl-beta-glucoside-solubilized state, our findings suggest that protein-protein interaction contributes to the stability of this protein.     

Enhancement of growth promoting activity of human blood on keratinocytes after its irradiation in vivo (transcutaneously) and in vitro with visible and infrared polarized light

To stimulate wound healing, current medicine uses various methods of phototherapy. The induced activation of proliferative processes in the wound occurs due to development of not only local, but also systemic processes, whose nature remains largely uninvestigated. The present work provides evidences that as early as 30 min after irradiation of a small area of the volunteer's body surface with polychromatic visible light + infrared polarized light (400-3400 nm, 95% of polarization) at a therapeutic dose (12 J/cm2), soluble factors appear in the circulating blood, which are able to stimulate proliferation of human keratinocytes in primary culture. A similar effect was also revealed after a direct blood irradiation. A proof is provided in favor of a hypothesis that a rapid rise of growth promoting activity of the entire circulating blood may be a consequence of transcutaneous photomodification of the small amount of light-modified blood in superficial skin vessels, and of the effect of such blood on its entire circulating volume. A possibility of a release into plasma of growth factors from blood cells and complexes with alpha 2-macroglobulin is discussed.     

Activation of the synthesis of RNA in lymphocytes following irradiation by a He-Ne-laser]

The rate of 14C-uridine incorporation during the first 12 hours after exposure of human peripheral blood lymphocytes to He-Ne laser radiation (lambda = 632.8 nm, D = 56 J/m2) has been determined. The stimulation of RNA synthesis is maximum 2 and 4 h following irradiation. The same regularity is noted after the addition of phytohemagglutinin (PHA). In 7 h the rate of RNA synthesis in irradiated cells is at the control level whereas in PHA stimulated cells the rate of 14C-uridine considerably increases.     

Studies in experimental animal models of the effect of ultraviolet radiation (UVC) on blood and isolated cell populations

The cellular and molecular basis of the therapeutically used effect of reinjected ultraviolet ( UVC ) irradiated blood is unknown. First approaches to that problem were made in this study by aid of model experiments. Neither the spontaneous degranulation nor the antigen-induced histamine release from rat connective tissue mast cells (in vivo) was influenced by the injection (i.v.) of ultraviolet irradiated blood or blood lymphocytes. By comparison of the effect of ultraviolet light on blood lymphocytes (number of dead cells, strength of chemoluminescence) after irradiation of the isolated cells and the unfractionated blood respectively, we could show that the strong light absorption within the blood sample prevents damage or functional alterations of the blood lymphocytes. The compound 48/80-induced histamine release from rat peritoneal mast cells can be completely inhibited by ultraviolet irradiation (0.6 mJ/cm2) without increasing the spontaneous histamine release.     

Mutagenic and lethal action of polychromatic near-ultraviolet (325-400 nm) on Haemophilus influenzae in the presence of nitrogen

The lethal effect of polychromatic near-UV light (325-400 nm) on Haemophilus influenzae was 8 times higher under aerobic than anaerobic irradiation. This light increased the frequency of mutation to novobiocin resistance and ability to utilize protoporphyrin IX. The slope of mutagenic effect at low doses appeared greater for the aerobic than for the anaerobic group. We concluded that polychromatic near-UV mutation of H. influenzae under anaerobic irradiation was caused by direct oxygen-independent action on DNA.     

A comparative study of chromatin from lymphocyte nuclei upon activation of transcription by irradiation from an He-Ne-laser or phytohemagglutinin]

The influence of He-Ne laser radiation (632.8 nm, 56 J/m2, t = 10 s) and phytohaemagglutinin (PHA, 2 micrograms/ml) on chromatin structure in human lymphocytes was studied by electron microscopy using ultrathin cell sections. Morphometric analysis of extranuclear condensed chromatin masses was performed 1 h after the irradiation or after the beginning of PHA treatment. In the irradiated cells the following insignificant changes were revealed: decrease in the relative area of the nucleoplasmic chromatin, increase in the relative area of decondensation zones as well as increase in the number of clumps of nucleoplasmic chromatin and relative length at their boundary with nucleoplasma. The tendency of these morphological changes may be interpreted as functional activation of extranucleolar RNA synthesis in response to irradiation by red laser light. Action of PHA results in significant changes of the surfaces of chromatin clumps, namely increase in relative length of nucleoplasmic chromatin boundary and decrease in relative length of perimembranous chromatin boundary with nucleoplasma as well as some less expressed delamination of the chromatin masses from the nuclear membrane. These essential changes may reflect chromatin activation by proliferative stimulus. Peculiarities of the ultrastructural reorganisation in the condensed chromatin after irradiation and PHA-treatment probably reflect the differences in the processes of gene activation caused by the two agents.     

Photomutagenicity of cosmetic ingredient chemicals azulene and guaiazulene

The photomutagenicity of the popular skin conditioning agents azulene and guaiazulene were tested in Salmonella typhimurium TA98, TA100 and TA102. Following irradiation with UVA and/or visible light, both azulene and guaiazulene exhibited mutagenicity 4-5-fold higher than the spontaneous background mutation. In contrary, naphthalene, a structural isomer of azulene, was not photomutagenic under the same conditions. Azulene was photomutagenic when irradiated with UVA light alone, visible light alone, or a combination of UVA and visible light. Azulene and guaiazulene are not mutagenic when the experiment is conducted with the exclusion of light. Therefore, extreme care must be taken when using cosmetic products with azulene/guaiazulene as ingredients since after applying these products on the skin, exposure to sunlight is inevitable.     

Oxidative stress by visible light irradiation suppresses immunoglobulin production in mouse spleen lymphocytes

In this study, we attempted to induce the oxidative stress in mouse spleen lymphocytes with visible light irradiation and examined the effects of lipid peroxidation on immunoglobulin (Ig) production. The spleen lymphocytes were isolated from 8-week-old male balb/c mice and irradiated with 300 W visible light. When the cells were cultured for 72 hr, Ig contents in culture supernatants were decreased gradually by irradiation for over 30 min. The cell viability was also lowered by the irradiation. Intracellular phosphatidylcholine hydroperoxide (PCOOH) levels and thiobarbituric acid-reactive substances (TBARS) values in culture supernatants were measured as indices of lipid peroxidation and we found that Ig production by mouse spleen lymphocytes was suppressed accompanied with the progress of peroxidation of intracellular phospholipids. Cell membrane fluidity was also significantly decreased, but the intracellular Ig level was not changed in the irradiated cells. These results suggest that the peroxidation of intracellular lipids is a cause of the suppression of Ig production by mouse spleen lymphocytes via lowering cell viability and suppressing Ig synthesis and secretion.     

The inhibition of mitosis in tissue culture cells by blue light

Blue light (wavelength 350-480 nm) irradiation of the early mitotic (prophase and prometaphase) tissue culture cells at the dose of 50-3000 J/cm2 delay mitosis or completely block it at the metaphase. Cell sensitivity to the near UV light (wavelength 360 nm) was few times more as compared with the sensitivity to the visible light (wavelength 400-480 nm). Mitotic cells irradiated with the green light (wavelength more than 500 nm; dose up to 7500 J/cm2) completed division normally. The effect of the blue light did not depend on the presence of phenol red in tissue culture medium. Rhodamin 123 staining did not show any changes in the mitochondrial system in the irradiated mitotic cells. Blue light irradiation with the dose enough for the induction of mitotic delay appears to be insufficient to affect the proliferation of interphase cells.     

X-irradiation of equine peripheral blood lymphocytes stimulated with phytohaemagglutinin in vitro.

Small lymphocytes were isolated from the peripheral blood of horses and incubated at 37 degrees C in Eagle's medium supplemented with 20 per cent foetal calf serum. The addition of phytohaemagglutinin (PHA) to the cultures resulted in: increased RNA and protein synthesis; the enlargement of the small lymphocyte into a lymphoblast-like cell; the initiation of DNA synthesis, and cell division. When survival was measured 24 hours after X-irradiation by means of phase-contrast microscopy, the lymphoblast-like cell was much more radio-resistant (D0 = 250 rad) than the small lymphocyte (D0 = 20 rad). This increase in radioresistance, however, was not observed until 12-24 hours after PHA treatment. To investigate which of the changes occurring during the transformation of the small lymphocyte was responsible for the increased resistance to irradiation, the percentage of cells surviving irradiation was compared with the percentage of cells incorporating significant amounts of 3HTdR, 3H-UR, or 3H-leucine at the time of irradiation. For this comparison, a dose of 100 rad was used because 100 rad killed essentially all of the small lymphocytes, but less than 35 percent of the cells which had become radioresistant from the PHA treatment. The results indicated that the increase in radioresistance was not associated with DNA synthesis, but instead correlated with the increase in RNA and protein synthesis which the cells had attained at the time of irradiation.     

Leukocyte numbers during the humoral and cell-mediated immune response of Japanese quail after microwave irradiation in ovo

1. Coturnix coturnix japonica eggs were exposed to 2.45-GHz continuous wave microwave radiation at an incident power density of 5 mW/cm2 (SAR = 4 mW/g) during the first 12 days of embryogeny. After hatching, leukocyte differential changes were measured in response to an injection with Alectoris graeca chukar red blood cells (CRBC) and in response to a phytohemagglutinin (PHA) injection in irradiated and nonirradiated (sham) quail of both sexes. 2. Microwave irradiation did not affect anti-CRBC hemagglutinin titers, PHA-evoked dermal swelling or leukocyte numbers and percentages. 3. In both the irradiated and sham irradiated males, lymphocyte percentages decreased while heterophil percentages increased after CRBC or PHA injection. 4. In ovo irradiation with microwaves did not alter the time course of either a humoral immune response or a cell-mediated immune response in Japanese quail.     

DNA Repair in Potorous tridactylus

The DNA synthesized shortly after ultraviolet (UV) irradiation of Potorous tridactylis (PtK) cells sediments more slowly in alkali than that made by nonirradiated cells. The size of the single-strand segments is approximately equal to the average distance between 1 or 2 cyclobutyl pyrimidine dimers in the parental DNA. These data support the notion that dimers are the photoproducts which interrupt normal DNA replication. Upon incubation of irradiated cells the small segments are enlarged to form high molecular weight DNA as in nonirradiated cells. DNA synthesized at long times (~ 24 h) after irradiation is made in segments approximately equal to those synthesized by nonirradiated cells, although only 10-15% of the dimers have been removed by excision repair. These data imply that dimers are not the lesions which initially interrupt normal DNA replication in irradiated cells. In an attempt to resolve these conflicting interpretations, PtK cells were exposed to photoreactivating light after irradiation and before pulse-labeling, since photoreactivation repair is specific for only one type of UV lesion. After 1 h of exposure ~ 35% of the pyrimidine dimers have been monomerized, and the reduction in the percentage of dimers correlates with an increased size for the DNA synthesized by irradiated cells. Therefore, we conclude that the dimers are the lesions which initially interrupt DNA replication in irradiated PtK cells. The monomerization of pyrimidine dimers correlates with a disappearance of repair endonuclease-sensitive sites, as measured in vivo immediately after 1 h of photoreactivation, indicating that some of the sites sensitive to the repair endonuclease (from Micrococcus luteus) are pyrimidine dimers. However, at 24 h after irradiation and 1 h of photoreactivation there are no endonuclease-sensitive sites, even though ~ 50% of the pyrimidine dimers remain in the DNA. These data indicate that not all pyrimidine dimers are accessible to the repair endonuclease. The observation that at long times after irradiation DNA is made in segments equal to those synthesized by nonirradiated cells although only a small percentage of the dimers have been removed suggests that an additional repair system alters dimers so that they no longer interrupt DNA replication.     

Assessment of ER Stress and Autophagy Induced by Ionizing Radiation in Both Radiotherapy Patients and Ex Vivo Irradiated Samples

Acute radiation leads to several toxic clinical states and triggers some molecular pathways. To shed light on molecular mechanisms triggered by ionizing radiation (IR), we examined the expression profiles of endoplasmic reticulum (ER) stress and autophagy‐related genes in individuals who were exposed to IR. Blood samples were collected from 50 cancer patients before radiotherapy and on the 5th, 15th, and 25th days of the treatment. Peripheral blood samples from 10 healthy volunteers were also obtained for ex vivo irradiation, divided into five and irradiated at a rate of 373 kGy/h to 0, 0.1, 0.5, 1, and 3Gy γ‐rays using a constant gamma source. GRP78, ATG5, LC3, ATF4, XBP1, and GADD153 genes were analyzed by quantitative real‐time polymerase chain reaction (QRT‐PCR) using beta 2 microglobulin (B2M) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) as references. In both groups, expressions of the selected genes have increased. It can be concluded that IR induces ER stress and related authophagy pathway in the peripheral lymphocyte cells proportionally by dose.