Effect of low-intensity laser radiation (632.8 nm) on immune cells isolated from mice

The effect of in vitro exposure to low-power laser light with a power density of 0.2 mW/cm2 and a wavelength of 632.8 nm induced by helium-neon laser on the functional activity of macrophages and splenic lymphocytes was studied. If the exposure period did not exceed 60 sec, the stimulation in interleukin-2 (IL-2) and nitric oxide (NO) production, as well as an increase in the activity of natural killer cells were observed. The increase of irradiation dose by prolongation of the exposure duration up to 180 s induced a significant decrease in NO production and natural killer cell activity, but IL-2 production was not different from the control level. A remarkable decrease in interferon-gamma (IFN-gamma) production was observed following laser light exposure of cells for 60 or 180 s, whereas under lower doses (exposure for 5 or 30 sec) IFN-gamma production increased. Irradiation of isolated macrophages induced a significant stimulation of cellular tumor necrosis factor-alpha (TNF- alpha) production at all dboes used, and, what is more important, an enhancement in both TNF-a phaand interleukin-6 (IL-6) production was revealed as early as after a 5-s exposure. In this case, more prolonged exposure periods, 60 and 180 s, either did not induce changes in IL-6 production (in macrophages) or decreased IL-6 production (in lymphocytes). Thus, upon in vitro exposure of cells to extremely low-power laser light, a basic tendency was observed: short-term irradiation predominantly induced stimulation in secretory activity of cells, whereas prolongation of exposure mainly induced immunosuppression. The only exception to the rule was a change in interleukin-3 (IL-3) production, which decreased after short-time exposure, but, on the opposite, increased when the cells were exposed for 180 s. In addition, a high sensitivity to extremely low-power laser light was supported by expression of the inducible heat shock protein, Hsp70, the effect being observed at all doses used, including the exposure for 5 s. At the same time, expression of another heat shock protein, Hsp90, was somewhat reduced after irradiation of cells with laser light.     

Effect of low intensity of electromagnetic radiation in the centimeter and millimeter range on proliferative and cytotoxic activity of murine spleen lymphocytes

It was found that single total-body exposure to electromagnetic centimeter waves (8.15-18 GHz, 1 microW/cm2, 5 h) stimulated the proliferation of mouse T and B splenic lymphocytes. The same effects were observed upon in vivo treatment of rats for 5 h with millimeter waves (42.2 GHz, amplitude modulation 10 Hz, 1 microW/cm2). The whole-body irradiation with centimeter or millimeter waves did not cause any significant changes in natural activity of killer cells. The cellular responses induced by the irradiation of isolated animal cells in vitro did not coincide with those revealed after the total-body irradiation of animals. Thus, the in vitro irradiation of natural killer cells to millimeter waves for 1 h increased their cytotoxic activity whereas, after treatment to centimeter waves for the same time, the activity of killer cells did not change. On the contrary, irradiation of T and B lymphocytes with millimeter waves (42.2 GHz, amplitude modulation 10 Hz, 1 microW/cm2, 1 h) suppressed the blasttransformation of cells. The results show a higher immunostimulative potential of centimeter waves as compared to millimeter waves.     

The effects of prolonged administration of 5-bromodeoxyuridine on cells of the immune system

We have determined the in vivo effect of 5-bromodeoxyuridine (BrdU) administered to mice in the drinking water for various lengths of time on the performance of T and B lymphocytes in a number of experimental protocols. Young mice continuously exposed to BrdU fail to gain weight, and the lymphocytes recovered after a prolonged period of exposure are fewer in number than in control mice. The recovery of normal levels of T and B lymphocytes after irradiation is severely impaired. Ag-specific cells responding to Ag in an adoptive transfer model fail to expand as much in the presence of BrdU as in the absence, and the Ag-specific effectors produced in the presence of BrdU are less able to secrete cytokines upon restimulation in vitro. Polarized populations of Tc1 and Tc2 effectors generated in vitro proliferate less in the presence of BrdU, and the resulting effectors make less cytokines per cell upon restimulation. Thus, the incorporation of BrdU into T or B lymphocytes can, under some circumstances, seriously impair the performance of the labeled cells, and these findings raise a note of caution in the interpretation of studies that make use of long-term exposure to BrdU.     

Effects of weak laser radiation (632.8 nm) on isolated mouse immune cells

The dose dependence of in vitro effects of low-intensity radiation of a He-Ne laser (632.8 nm, 0.2 mW/cm²) on the functional activity of peritoneal macrophages and lymphocytes of mouse spleen was studied. The exposure of isolated cells was varied from 5 to 180 s. If the exposure did not exceed 60 s, stimulation of secretory activity was observed: increased production of interleukin 2, interferon γ, and interleukin 6 in lymphocytes; increased production of tumor necrosis factor α, nitric oxide, and interleukin 6 in macrophages; and enhanced activity of natural killer cells. A longer exposure (up to 180 s) either had no effect on the synthesis of certain cytokines (interleukin 2 in lymphocytes and interleukin 6 in macrophages) or inhibited it, which was expressed in decreased production of interleukin 6 and interferon γ in lymphocytes and nitric oxide in macrophages, as well as in suppression of the activity of natural killer cells. Conversely, the production of interleukin 3 decreased after a short-term exposure but increased after 180-s irradiation. The high sensitivity of cells to extremely weak laser light also manifested itself as a considerable increase in expression of the inducible heat shock protein 70; this effect was observed at all doses studied, including the 5-s exposure. In contrast, expression of the heat shock protein 90 slightly decreased after irradiation of cells with laser light.     

The cytokinesis-block micronucleus assay as a biological dosimeter in spleen and peripheral blood lymphocytes of the mouse following acute whole-body irradiation

To evaluate the application of the cytokinesis-block (CB) micronucleus (MN) assay as a biological dosimeter following in vivo exposure to ionising radiation we determined the micronucleus frequency in spleen and peripheral blood lymphocytes of the mouse, serially, for 14 days following acute whole-body irradiation. The baseline MN frequency of spleen lymphocytes (7.86 +/- 0.68, mean +/- 1 SD) was significantly (p less than 0.001) elevated when compared to that for peripheral blood lymphocytes (4.10 +/- 0.53). Immediately after irradiation there was a substantial dose-related increase in MN, but the MN frequencies in spleen lymphocytes (120.2 +/- 9.4 for 1 Gy; 409.5 +/- 38.4 for 2 Gy) were significantly (p less than 0.009) elevated compared to those in peripheral blood lymphocytes (78.0 +/- 7.0 for 1 Gy; 200.2 +/- 10.9 for 2 Gy). During the 14 days after irradiation, the MN frequency in spleen lymphocytes declined gradually to approximately half of the value observed immediately after irradiation. By contrast the MN frequency in peripheral blood lymphocytes increased during the week after irradiation, but ultimately MN frequencies in blood and spleen became approximately the same by day 14. Study of isolated murine lymphocytes irradiated in vitro showed that the number of MN generated by a given dose of radiation was approximately 2-3 times greater than the number generated by in vivo irradiation. These results suggest that measurement of MN in vivo after irradiation can be used as an in vivo dosimeter. However, precise dosimetry is probably affected by factors such as kinetic changes in different lymphocyte populations and possibly by in vivo factors which influence sensitivity of cells to radiation.     

Activity of dsRNA-dependent protein kinase in rat lymphoid cells under the effect of X-ray irradiation

The activity of dsRNA-dependent protein kinase, which is the key enzyme of the interferon signal system, was studied in the rat spleen and thymus lymphocytes under the influence of X-ray irradiation at 0.5 and 1 Gy doses and interferon inducers administration. An increase of the enzyme activity was established in the presence of FGA, concanavaline A, poly(I).poly(C) in vitro. The effect is intensified under the irradiation by 0.5 Gy dose. The protein kinase activity in lymphocytes is amplified in proportion to poly(I).poly(C) concentration, that was most pronounced in the irradiated animals. The comparative analysis of the action of interferon inducers on the dsRNA-dependent protein kinase activity was carried out. Two biological systems were used: in vivo (when the preparations were injected to the experimental animals) and in vivo (under the preincubation of isolated lymphocytes with the inducers). It was shown that the combined action of radiation and interferon inducers causes the stimulation of dsRNA-dependent protein kinase activity.     

The effect of recombinant human interleukin-1 beta on the repopulation of bone-marrow colony-forming cells in mice subjected to the action of radiation and burns]

The radioprotective and restorative (therapeutic) effects of human recombinant interleukin-1 beta (IL-1 beta) on the population of bone marrow CFU-S of mice, subjected to either sublethal doses of ionising irradiation itself or the same irradiation in combination with thermal burn, are investigated. Both the effects of the agent are registered under both in vitro and in vivo irradiation in semi-, syn- and allogeneic animals. If the irradiation was combined with thermal burn, the "therapeutic" effect of the agent was demonstrated at irradiation dose equal to 3.06 Gy rather than to 6.12 Gy. If the bone marrow cells were irradiated in vitro in dose 3.06 Gy with the following heat shock at 42 degrees C for 10-20 min, the "therapeutic" effect of IL-1 beta was seen only if it was added to cells before rather than after irradiation. The radioprotective effect of IL-1 beta is maintained under in vitro, as well as in vivo conditions in the allogeneic system of transplantation of the CBA donor bone marrow to the C57BL mice.     

Lymphocyte subset analyses in blood, spleen and lymph nodes of female Sprague-Dawley rats after short or prolonged exposure to a 50 Hz 100-microT magnetic field.

Based primarily on the results of in vitro studies, it has been suggested that power-line (50 or 60 Hz) magnetic fields (MFs) may reduce immune function, which could lower resistance to infection or cancer. This study was conducted to evaluate the influence of acute and chronic in vivo exposure to a linearly polarized 50 Hz MF on immune function in female Sprague-Dawley rats. Groups of rats were exposed continuously to the MF at a flux density of 100 microT for periods of 3 days, 14 days or 13 weeks. For each exposure period, one control group of rats was sham-exposed together with each MF-exposed group. Experimental end points included analyses of T-lymphocyte subsets as well as other immune cells involved in cell-mediated immune responses, i.e. natural killer (NK) cells, B lymphocytes, macrophages, and granulocytes in blood, spleen and mesenteric lymph nodes. In addition, immunohistochemical methods were used to detect proliferating and apoptotic cells in the various compartments of spleen tissue. The results obtained failed to demonstrate a significant effect of short or prolonged MF exposure on different types of leukocytes, including lymphocyte subsets. Furthermore, the experiments on the in vivo proliferation activity of lymphocytes and the extent of apoptosis in spleen samples did not indicate a difference between the MF-exposed and sham-exposed groups, indicating that MF exposure does not affect the mechanisms involved in the control of lymphocyte homeostasis. The lack of MF effects in the immune tests used in the present in vivo study makes it highly unlikely that MF exposure induces immunotoxicity, at least under the experimental conditions used. However, the data do not exclude the possibility that functional alterations in T-cell responses to mitogens and in NK cell activity as recently described for MF-exposed rodents may be one mechanism involved in the carcinogenic effects of MF exposure observed in some models of co-carcinogenesis.     

Proliferative activity of lymphocyte cultures due to the mutagenic action of thiophosphamide in vivo and in vitro

Thiophosphamide is capable of inhibiting the cell cycle after the treatment of rabbit blood lymphocytes in vivo and in vitro. To assess the proliferative activity of the cells during cultivation, use was made of the index of the mean division number which was experienced by the cells after mutagenic exposure prior to fixation. As the mutagen dose was raised in vivo and in vitro, the mean division number decreased linearly, namely by approximately the same value with an equivalent dose variation. This indicates that thiophosphamide produces the same cytotoxic action both in vivo and in vitro.     

Stimulation of murine natural killer cells by weak electromagnetic waves in the centimeter range]

Irradiation with electromagnetic waves (8.15-18 GHz, 1 Hz within, 1 microW/cm2) in vivo increases the cytotoxic activity of natural killer cells of rat spleen. In mice exposed for 24-72 h, the activity of natural killer cells increased by 130-150%, the increased level of activity persisting within 24 h after the cessation of treatment. Microwave irradiation of animals in vivo for 3.5 and 5 h, and a short exposure of splenic cells in vitro did not affect the activity of natural killer cells.     

UVB induced oxidative stress in human keratinocytes and protective effect of antioxidant agents

This study aims at exploring the oxidative stress in keratinocytes induced by UVB irradiation and the protective effect of nutritional antioxidants. Cultured Colo-16 cells were exposed to UVB in vitro followed by measurement of reactive oxygen species (ROS), endogenous antioxidant enzyme activity, as well as cell death in the presence or absence of supplementation with vitamin C, vitamin E, or Ginsenoside Panoxatriol. Intracellular ROS content was found significantly reduced 1 h after exposure, but increased at later time points. After exposure to 150–600 J m⁻² UVB, reduction of ROS content was accompanied by increased activity of catalase and CuZn-superoxide dismutase at early time points. Vitamins C and E, and Ginsenoside Panoxatriol counteracted the increase of ROS in the Colo-16 cells induced by acute UVB irradiation. At the same time, Ginsenoside Panoxatriol protected the activity of CuZn-superoxide dismutase, while vitamin E showed only a moderate protective role. Vitamins C and E, and Ginsenoside Panoxatriol in combination protected the Colo-16 cells from UVB-induced apoptosis, but not necrosis. These findings suggest that vitamins C and E as well as Ginsenoside Panoxatriol are promising protective agents against UVB-induced damage in skin cells.     

Generation of reactive oxygen species and radiation response in lymphocytes and tumor cells

Several types of lymphoid and myeloid tumor cells are known to be relatively resistant to radiation-induced apoptosis compared to normal lymphocytes. The intracellular generation of reactive oxygen species was measured in irradiated spleen cells from C57BL/6 and BALB/c mice and murine tumor cells (EL-4 and P388) by flow cytometry using dichlorodihydrofluoresceindiacetate and dihydrorhodamine 123 as fluorescent probes. The amount of reactive oxygen species generated per cell was low in the tumor cells compared to spleen cells exposed to 1 to 10 Gy of gamma radiation. This could be due to the higher total antioxidant levels in tumor cells compared to normal cells. Further, the changes in mitochondrial membrane potential and cytoplasmic Ca2+ content were appreciable in lymphocytes even at a dose of 1 Gy. In EL-4 cells, no such changes were observed at any of the doses used. About 65% of spleen cells underwent apoptosis 24 h after 1 Gy irradiation. However, under the same conditions, EL-4 and P388 cells failed to undergo apoptosis, but they accumulated in G2/M phase. Thus the intrinsic radioresistance of tumor cells may be due to a decreased generation of reactive oxygen species after irradiation and down-regulation of the subsequent events leading to apoptosis.     

Apoptosis development pathways in human lymphocytes induced by UV light and reactive oxygen species

We have studied alterations in the structural state of DNA, the level of membrane Fas-receptor expression, functional activity of caspase-3, the concentration of Ca²⁺, p53 and cytochrome c proteins in human lymphocyte cells in the dynamics of apoptosis, induced by UV light (240–390 nm) at doses of 151, 1510, and 3020 J/m² and reactive oxygen species (ROS): superoxide anion radical, hydroxyl radical, hydrogen peroxide, and singlet oxygen. It was established that UV light and ROS induce lymphocyte DNA fragmentation after the incubation of a modified cell for 20 h. It was shown that in 1–5 h after UV light and ROS exposure on lymphocytes, an increase is observed in the level of membrane death Fas-receptors as compared to intact cells. Enhancement was revealed in the functional activity of lymphocyte caspase-3 4 h after the generation of singlet oxygen, hydroxyl radical, and the addition of hydrogen peroxide, as well as 8 and 24 h and 6 and 8 h of UV irradiation of cells at doses of 151 and 1510 J/m², respectively. Using the DNA comet approach, it was revealed that DNA damage (single-stranded breaks) appears approximately 15–20 min after UV irradiation of lymphocytes at doses of 1510 and 3020 J/m² and the addition of hydrogen peroxide at a concentration of 10⁻⁶ mol/L (comets of the C1 type) and reaches its maximum 6 h after cell modification (comets of the C2 and C3 types). Six hours after exposure of lymphocytes to hydrogen peroxide and UV light at doses of 1510 and 3020 J/m², it was established that the p53 level increased in the investigated cells. It was established that under UV light exposure and exogenous generation of reactive oxygen species, the increase in the calcium level in lymphocyte cytoplasm is determined by Ca²⁺ efflux from the intracellular depots as a result of activation of the components of the phosphoinositide information transmission mechanism to a cell. A hypothesis was proposed on the correlation between changes in the calcium level and initiation of programmed cell death in human lymphocytes after UV light and ROS exposure. It was concluded that the lead role is played by receptor-mediated (Fas-dependent) caspase and p53-dependent pathways in the development of lymphocyte apoptosis induced by exposure to UV light at doses of 151 and 1510 J/m² and reactive oxygen metabolites. A scheme is presented which considers possible intracellular events leading to apoptotic death of lymphocytes after UV irradiation.     

In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.     

Antioxidant vitamins C, E and beta-carotene reduce DNA damage before as well as after gamma-ray irradiation of human lymphocytes in vitro

The protective effect of Vitamins C, E and beta-carotene against gamma-ray-induced DNA damage in human lymphocytes in vitro was investigated. Cultured lymphocytes were exposed to increasing concentration of these vitamins either before or after irradiation with 2Gy of gamma-rays and DNA damage was estimated using micronucleus assay. A radioprotective effect was observed when antioxidant vitamins were added to cultured cells before as well after irradiation; the strongest effect was observed when they were added no later than 1h after irradiation. The radioprotective effect of vitamins also depended on their concentration; Vitamins C added at low concentration (1 microg/ml) before exposure of the cells to radiation prevented induction of micronuclei. Vitamin E at the concentration above 2 microg/ml decreased the level of radiation-induced micronuclei when compared to the cells irradiated without vitamin treatment. beta-Carotene was effective at all tested concentrations from 1 to 5 microg/ml and reduced the number of micronuclei in irradiated cells. The vitamins had no effect on radiation-induced cytotoxicity as measured by nuclear division index. The radioprotective action of antioxidant Vitamins C, E and beta-carotene was dependent upon their concentration as well as time and sequence of application.     

Effects of in vivo ultrasound hyperthermia on natural killer cell cytotoxicity in the hamster

The effects of in vivo ultrasound irradiation of the spleen on immunological functions were assessed with an in vitro natural killer (NK) cell cytotoxic assay. Anesthetized hamsters were exposed to 1 MHz ultrasound at intensity levels currently being used clinically for therapeutic diathermy and hyperthermia (1-5 W/cm2, for 500 sec with constant beam scanning). Hyperthermic levels in the spleen ranged from 38-43 degrees C. Significant depression of natural killer (NK) cell activity was seen 4 h after spleen irradiation as compared to sham irradiated and normal animals. A return towards normal levels was observed in experimental groups at 24 h after exposure. Sham and normal animals were not significantly different in NK activity, indicating no significant stress-related immunosuppressive effects due to handling. Differential leukocyte counts taken for each exposure condition showed significant lymphopenia at 4, 8, and 16 h after exposure, near normal levels at 24 h, and complete recovery by 48 h. The number of circulating mononuclear cells at 4 h showed a dose-related suppression as the exposure intensities were increased.     

Comparison of the cytogenetic effects of cyclophosphamide on monkey lymphocytes in vivo and in vitro

The comparative in vivo and in vitro study of chromosomal aberrations and SCE induced by cyclophosphamide (CP) in macaca rhesus lymphocytes was performed. The dose of mutagenic exposure for quantitative estimation of effects was determined as a product of concentration of alkylating CP metabolites on the exposure time. The mutagenic effect caused by the same doses of CP (CP metabolites) appeared similar in vivo and in vitro. This suggests that the results obtained in adequate in vitro mutagen-testing experiments may be quantitatively extrapolated for the in vivo conditions.     

Lymphocyte cytochrome c oxidase, cyclic GMP and cholinergic muscarinic receptors as peripheral indicators of carbon monoxide neurotoxicity after acute and repeated exposure in the rat

Changes in cerebral cytochrome oxidase (COX) activity, nitric oxide (NO)-cyclic GMP (cGMP) pathway and cholinergic muscarinic receptors (MRs) have been reported in rodents acutely exposed to carbon monoxide (CO). These endpoints measurable in lymphocytes may serve as peripheral markers of CO neurotoxicity. The early and delayed effects of repeated and acute in vivo CO inhalation were investigated on COX activity, cGMP formation and MR binding in rat brain and lymphocytes to assess whether each endpoint was similarly affected both centrally and peripherally. Male Wistar rats either inhaled 500 ppm CO, 6 h/day, 5 days/week, 4 weeks (repeated exposure) or 2,400 ppm, 1 h (single exposure). Neither treatment altered brain or lymphocyte COX activity 1 and 7 days post-treatment. Also ineffective were repeated and acute CO treatments towards (3)H-quinuclidinyl benzilate (QNB) binding to MRs in cerebral cortex, hippocampus, striatum, cerebellum (respective controls, mean+/-S.D.: 171 +/- 45, 245 +/- 53, 263 +/- 14 and 77 +/- 7 fmol/mg protein) and lymphocytes (24 +/- 10 fmol/million cells) at the same time points. In lymphocytes control cGMP levels averaged 1.98 +/- 0.99 pmol/mg protein under basal conditions, and 3.94 +/- 0.55 pmol/mg protein after NO-stimulation. One day after chronic treatment cessation, the CO-treated group displayed about a 50% decrease in both basal and NO-stimulated cGMP values, which persisted up to 7 days after, compared to air-exposed rats. Acutely, CO caused a delayed enhancement (+140%) of NO-induced activation of soluble guanylate cyclase. The finding that the NO-cGMP pathway is a target for the delayed effects of CO in peripheral blood cells is in accordance with our data in brain [Hernández-Viadel, M., Castoldi, A.F., Coccini, T., Manzo, L., Erceg, S., Felipo, V., 2004. In vivo exposure to carbon monoxide causes delayed impairment of activation of soluble guanylate cyclase by nitric oxide in rat brain cortex and cerebellum. Journal of Neurochemistry 89, 1,157-1,165], and supports the use of this peripheral endpoint as a biomarker of CO central effects.     

Photo-chemically induced DNA effects in the comet assay with epidermal cells of SKH-1 mice after a single oral administration of different fluoroquinolones and 8-methoxypsoralen in combination with exposure to UVA

Due to the need for in vivo photo-genotoxicity tests, the in vivo photo-comet assay was established in epidermal cells of the SKH-1 mouse. Groups of 10 male SKH-1 mice each were treated once orally with vehicle only, with three fluoroquinolones (25 mg/kg clinafloxacin, 20 mg/kg lomefloxacin, 200 mg/kg ciprofloxacin) or with 200mg/kg 8-methoxypsoralene (8-MOP). Thirty minutes after treatment half of the mice in each group were exposed to 23.8 J/cm2 UVA. Thereafter the mice were killed and their epidermal cells tested in the alkaline (pH >13) comet assay; at the same time after administration, compound-treated, non-irradiated mice were killed and analysed. A negative control group of ten male SKH-1 mice received the vehicle only; half of these animals were exposed to UVA, half were not. The comet tail lengths of epidermal cells of the mice were statistically significantly increased for all three fluoroquinolones (FQ) tested in combination with UV irradiation. Treatment with 8-methoxypsoralene+UV induced a significant reduction of comet tail length. Tail intensity and tail moment gave essentially the same results after combined exposure (compound+UV). Without irradiation, the tail lengths of controls and compound-treated mice were comparable under the conditions of this study. In contrast, tail intensity and tail moment were increased for all test compounds (including 8-MOP), without irradiation. Irradiated controls had a tail length comparable to non-irradiated controls, while tail intensity and tail moment were clearly increased in irradiated controls. In conclusion: under the present experimental conditions the in vivo photo-comet assay is able to detect photo-chemically induced DNA strand breaks as well as photo-chemically induced DNA cross-links.