Aloe vera modulates X-ray induced hematological and splenic tissue damage in mice.

The present study was premeditated to examine the radioprotective effects of aqueous Aloe vera gel extract against whole-body X-ray irradiation-induced hematological alterations and splenic tissue injury in mice. Healthy male balb/c mice were divided into four groups: group 1, control; group 2, A. vera (50 mg/kg body weight) administered per oral on alternate days for 30 days (15 times); group 3, X-ray exposure of 2 Gy (0.25 Gy twice a day for four consecutive days in the last week of the experimental protocol); and group 4, A. vera + X-ray. X-ray exposure caused alterations in histoarchitecture of spleen along with enhanced clastogenic damage as assessed by micronucleus formation and apoptotic index. Irradiation caused an elevation in proinflammatory cytokines like tumor necrosis factor and interleukin-6, total leucocyte counts, neutrophil counts and decreased platelet counts along with unaltered red blood cell counts and hemoglobin. Irradiation also caused an elevation in reactive oxygen species (ROS), lipid peroxidation (LPO) levels, lactate dehydrogenase activity and alterations in enzymatic and nonenzymatic antioxidant defense mechanism in plasma and spleen. However, administration of A. vera gel extract ameliorated X-ray irradiation-induced elevation in ROS/LPO levels, histopathological and clastogenic damage. It also modulated biochemical indices, inflammatory markers, and hematological parameters. These results collectively indicated that the A. vera gel extract offers protection against whole-body X-ray exposure by virtue of its antioxidant, anti-inflammatory and anti-apoptotic potential.

Radioprotective effect of grape seed extract against gamma irradiation in mouse bone marrow cells.

INTRODUCTION: Ionizing radiations produce free radicals which are often responsible for DNA damage or cell death. Grape seed extract (GSE) is a natural compound having an antioxidant that protects DNA, lipids, and proteins from free radical damages. In this study, radioprotective effect of the GSE has been investigated in mouse bone marrow cells using micronucleus test. MATERIALS AND METHODS: Four groups of mice were investigated in this study: Mice in Group 1 were subjected to injection of distilled water with no irradiation. Mice in Group 2 were exposed to 3 Gy gamma radiation after the injection of distillated water. Mice in Group 3 were injected with 200 mg/kg of the GSE without any irradiation. In another group, mice were exposed to three gray gamma irradiation after the injection of GSE. Animals were killed, and slides were prepared from the bone marrow cells 24 h after irradiation. The slides were stained with May Grunwald-Giemsa method and analyzed microscopically. The frequency of the micronucleated polychromatic erythrocytes (MnPCEs), micronucleated normochromatic erythrocyte (MnNCEs), and polychromatic erythrocyte/polychromatic erythrocyte + normochromatic erythrocyte (PCE/PCE + NCE) ratios was calculated. RESULTS: Injection of GSE significantly decreased the frequency of MnPCEs (P < 0.0001) and MnNCEs (P < 0.05) and increased the ratio of PCE/PCE + NCE (P < 0.0001) compared to the irradiated control group. DISCUSSION AND CONCLUSIONS: GSE could reduce clastogenic and cytotoxic effects of gamma irradiation in mice bone marrow cells; therefore, it can be concluded that the GSE is a herbal compound with radioprotective effects against gamma irradiation. Free radical scavenging and the antioxidant effects of the GSE probably are responsible mechanisms for the GSE radioprotective effects.

Intake of saffron reduces γ-radiation-induced genotoxicity and oxidative stress in mice.

Saffron (SAF), the dried stigmas of Crocus sativus, is commonly used for flavoring and coloring food. Studies on bioactivity of SAF have demonstrated its in vivo antioxidant activity. The aim of our study was to assess the impact of SAF intake on γ-radiation (RAD) induced (a) chromosomal damage, (b) oxidative stress in liver and brain, and (c) histopathological effects in the intestinal cells and male germ cells in mice. Freeze-dried aqueous extract of SAF was used for the experiments. Our preliminary cell-free DNA nicking assay using pBR322 DNA revealed protective effects of freeze-dried SAF extract against hydroxyl radical induced DNA damage. For the in vivo investigations, freeze-dried SAF extract in distilled water was administered by gavage (40 mg/kg b.w.) to male Swiss albino mice for six consecutive days. On the sixth day, the animals were exposed to RAD (1 or 2 Gy) and sacrificed 24 h later to collect bone marrow cells for assessing chromosomal damage by measuring micronucleated polychromatic erythrocytes (MnPCEs). Liver and brain samples from animals exposed to 2 Gy RAD were used for evaluating lipid peroxidation and activity of antioxidant enzymes. The testis and intestine were used for histopathological analysis. Our results demonstrated significant protective effects of SAF against RAD-induced genotoxic damage. SAF pretreatment reduced the level of lipid peroxidation with concomitant increase in glutathione content and activity of glutathione S-transferase, glutathione peroxidase, and catalase. The histopathological analysis showed minimal impact of SAF on RAD-induced damage in the intestinal cells and male germ cells.

Radioprotective effect of chicory seeds against genotoxicity induced by ionizing radiation in human normal lymphocytes.

The search for less-toxic radioprotective agents has led to a growing trend towards natural products. Protective effect of the methanolic extract of chicory seeds (MCS) was investigated against genotoxicity induced by ionizing radiation in human lymphocytes. Human peripheral blood samples were collected and incubated with MCS at different concentrations (10, 50, 100, and 200 µg/mL) for two hours. The whole blood samples were exposed in vitro to X-ray at dose 2.5 Gy. Then, the lymphocytes were cultured with mitogenic stimulation to determine the micronucleus in cytokinesis blocked binucleated cell. The methanolic extract at all doses significantly reduced the frequency of micronuclei in binucleated lymphocytes, as compared with similarly irradiated lymphocytes without any extract treatment. The maximum protection was observed at 200 µg/mL of MCS, it completely protected genotoxicity induced by ionizing radiation in human lymphocytes. The extract exhibited a concentration-dependent radical scavenging activity on 1,1-diphenyl-2-picryl hydrazyl free radicals. HPLC analysis of MCS showed this extract is containing chlorogenic acid as a phenolic compound. These data suggest that the radioprotective effect of methanolic extract of chicory seeds can be attributed to the presence of phenolic compounds such as chlorogenic acid which act as antioxidant agents.

Micronucleated erythrocytes in newborns of rat dams exposed to ultraviolet-A light during pregnancy; protection by ascorbic acid supplementation.

Pregnant hairless rat dams were exposed to ultraviolet-A light (UVA) to induce micronucleated erythrocytes (MNE) in their fetuses. The control group was exposed to conventional light; the experimental groups were exposed to UVA (365nm) during gestational days 16-21. In some cases, ascorbic acid (Asc) was administered in the drinking water from gestational day 15 until delivery. Dams were sampled at 48-h intervals during gestation, from day 16 until delivery. Blood was also obtained from neonates at birth; MNE, micronucleated polychromatic erythrocytes (MNPCE), and polychromatic erythrocytes (PCE) were scored. Increased MNE and MNPCE were observed in neonates born to mothers exposed to UVA for 40, 80 or 160min, compared to the control group. Asc treatment reduced MNE and MNPCE induction.

Influence of selenium on radiogenic collagen destruction and the degree of collagen tissue maturation in stage III oral squamous cell carcinoma patients undergoing therapeutic irradiation.

INTRODUCTION: We set out to assess whether selenium, an antioxidant mineral could influence radiogenic collagen maturation. MATERIALS AND METHODS: The study comprise of normal (Group I), untreated oral carcinoma cases (Group II) (n = 20), cases who underwent radiotherapy (Group IIa) n = 10 and cases supplemented with selenium along with radiotherapy (Group IIb) n = 10. RESULTS: Spectrophotometric estimation and luminescence spectral assignment of collagen showed improved collagen maturation status. Measurement of the mature collagen cross-links hydroxylysylpyridinoline and lysylpyridinoline by high-performance liquid chromatography on irradiated tissues showed a considerable decrease in the selenium Group IIb (P < 0.05) indicating a decrease in collagen fragments. Electron microscopic studies showed significant morphological alteration in the selenium group. The micro nucleus frequency, decreased in radiation group (P < 0.05) compared with untreated (P < 0.05). While much more decrease observed in the selenium group (P < 0.05). DISCUSSION: The results represent the effect of selenium treatment with a bearing on carcinogenic process to curtail it, thus enhancing the maturity of collagen.

Individual variations in the micronucleus assay for biological dosimetry after high dose exposure.

The micronucleus assay is widely used as a biological dosimeter. Due to an inhibitory effect of radiation on cell proliferation the assay yields satisfactory results only when the absorbed dose is below about 5Gy. In 2002 Müller and Rode suggested that a modified version of the test, based on the analysis of the ratio of trinucleated to tetranucleated cells and the frequency of micronuclei (Mn) in binucleated cells containing at least one Mn, can be applied to detect a dose reaching 15Gy (Mutat. Res. 502 (2002) 47-51). Their conclusion was based on the results of experiments with lymphocytes from one donor and nothing is known about the possible influence of individual variability on the applicability of the Mn test to detect high doses of radiation. The aim of the present study was to validate the modified micronucleus assay with lymphocytes of 5 donors. Their blood was exposed to 0, 5, 10, 15 and 20Gy of (60)Co gamma rays. The levels of Mn and of cell proliferation were assessed using various approaches. A strong inter-individual variability was observed for all endpoints. The results clearly show that the assessment of cell proliferation is essential for the interpretation of results. Unfortunately, it was not possible to identify one single proliferation marker that gives all necessary information.

Comparison of cytotoxic and genotoxic effects of plutonium-239 alpha particles and mobile phone GSM 900 radiation in the Allium cepa test.

The goal of this study was to compare the cytotoxic and genotoxic effects of plutonium-239 alpha particles and GSM 900 modulated mobile phone (model Sony Ericsson K550i) radiation in the Allium cepa test. Three groups of bulbs were exposed to mobile phone radiation during 0 (sham), 3 and 9h. A positive control group was treated during 20min with plutonium-239 alpha-radiation. Mitotic abnormalities, chromosome aberrations, micronuclei and mitotic index were analyzed. Exposure to alpha-radiation from plutonium-239 and exposure to modulated radiation from mobile phone during 3 and 9h significantly increased the mitotic index. GSM 900 mobile phone radiation as well as alpha-radiation from plutonium-239 induced both clastogenic and aneugenic effects. However, the aneugenic activity of mobile phone radiation was more pronounced. After 9h of exposure to mobile phone radiation, polyploid cells, three-groups metaphases, amitoses and some unspecified abnormalities were detected, which were not registered in the other experimental groups. Importantly, GSM 900 mobile phone radiation increased the mitotic index, the frequency of mitotic and chromosome abnormalities, and the micronucleus frequency in a time-dependent manner. Due to its sensitivity, the A. cepa test can be recommended as a useful cytogenetic assay to assess cytotoxic and genotoxic effects of radiofrequency electromagnetic fields.

Persistence of genetic damage in lymphocytes from former uranium miners.

Lymphocytes from former uranium miners who finished work underground one or more decades ago were analysed with respect to possibly persisting genetic damage induced by their radiation exposure. A modified micronucleus-centromere test was used which determined the frequency of micronucleus-containing binucleate cells after cytochalasin B treatment and the percentage of centromere-free micronuclei, assessed with the help of immunofluorescence labeling of centromere protein B. Whereas the overall frequency of micronucleus-containing cells was not significantly elevated above the level found in a control group, former miners showed a greater percentage of centromere-free micronuclei, i.e. micronuclei containing only acentric fragments. Our results are in excellent agreement with those of an earlier uranium miner study and lend support to the assumption that genetic damage from alpha radiation can persist for many years after exposure, possibly due to genomic instability. The frequency of micronucleus-containing cells, but not the percentage of centromere-free micronuclei, significantly increased with time since last exposure in the mines. This can be attributed, at least in part, to the fact that miners who have finished working underground longer ago tend to be older, and there is an increase of the frequency of micronucleus-containing cells with age.

Micronuclei in lymphocytes from currently active uranium miners.

Micronuclei can be used as markers of past radiation exposure, but only few studies have dealt with uranium miners. In this paper, we report on micronuclei in lymphocytes from individuals currently working at Rozná, Czech Republic, the last functioning uranium mine in the European Union. A modified micronucleus-centromere test was applied to assess the occurrence of micronuclei in stimulated lymphocytes, as well as their content in terms of whole chromosomes or fragments. Compared with unexposed individuals, the miners had higher frequencies of micronucleus-containing lymphocytes and higher percentages of micronuclei without centromeres, and the differences were significant for both parameters (0.74 ± 0.60 vs. 0.50 ± 0.42, p = 0.017 and 49 ± 44 vs. 12 ± 21, p = 0.0002; means ± standard deviations). There were also significant correlations between one or other of these parameters on the one hand and various dose values on the other, in particular with a 'retrievable' dose, that is, a dose whose effect should still be recognisable in lymphocytes assuming a half-life of 3 years. The 'retrievable' dose at which a doubling of the micronucleus frequency was observed was around 35 mSv, corresponding to a total dose of 90 mSv received while working in the mines. Altogether, our data show that the micronucleus-centromere test is a valuable tool for the assessment of past radiation exposure in uranium miners. The scatter in the data is of course far too great to allow individual dosimetry, but for groups of a few dozen exposed individuals, the method can be used to monitor doses clearly below 100 mSv.

Increased frequency of micronuclei in lymphocytes of infertile males after exposure to gamma irradiation: a possible sign of genomic instability.

PURPOSE: Induced chromosomal instability and micronucleus (MN) formation in blood lymphocytes of infertile men in comparison with fertile men exposed to gamma radiation was investigated. METHODS: Blood samples of healthy and infertile donors were irradiated by 2 and 4 Gy Co-60 gamma-rays, then cultured in RPMI-1640 complete medium containing 1% phytoheamaglutinin (PHA) and incubated in a CO(2) incubator. Cytochalasin-B was added to the cultures at a final concentration of 4 µg/ml. Finally, harvesting, slide making, and analysis were performed according to standard procedures. RESULTS: We observed a statistically significant difference between the frequencies of micronuclei in lymphocytes of infertile individuals, compared to healthy donors, before and after exposure to gamma rays. Although higher in azoospermia patients, the frequency of MN was not statistically different between infertile groups. CONCLUSIONS: This study indicates that genomic instability in infertile men could probably contribute to the development of an impaired reproductive capacity.

Transient cytotoxicity of ¹³¹I beta radiation in hyperthyroid patients treated with radioactive iodine.

BACKGROUND & OBJECTIVES: Radioiodine ( 131 I) or radioactive iodine in low doses is used worldwide as the first line of management in the treatment of hyperthyroidism. Information is available on the extent and severity of cell damage after a high dose radioiodine ( 131 I) therapy for thyroid cancer, but information is scanty on its cellular effects, its extent and severity of cell damage after a low dose 131 I therapy. The present investigation was aimed to study the cytotoxic effects of a low dose 131 I therapy in varying doses as is normally being used in routine clinical practice in the treatment of various forms of hyperthyroidism. METHODS: Peripheral blood lymphocytes were analyzed in 32 hyperthyroid patients. All of them received 131 I in the form of sodium iodide solution orally. Blood lymphocytes were studied for the presence of chromosomal aberrations (CA) and micro nucleus (MN) using micronucleus assay. Blood samples of these patients were drawn prior to the treatment, on 7 th and 30 th days after the treatment. RESULTS: The results indicated a positive relationship between 131 I dose, CA and MN frequency. A statistically significant increase in CA and MN frequency in day 7 post- therapy and a decrease in mean levels of CA and MN on day 30 post-therapy were observed when compared to pre-therapy. INTERPRETATION & CONCLUSIONS: This study showed that the cytogenetic damage induced by 131 I in low doses i.e., less than 555MBq was minimal and reversible. Patients can be motivated to undertake this safe and easy procedure as a first line of therapy in the treatment of hyperthyroidism.

[Effect of radio-ecological conditions in the ChNPP exclusion zone on morphofunctional state and responsiveness of organs and tissues of laboratory animals].

The state of hematopoietic, reproductive and endocrine systems of the organisms of male rats and their offspring in generations (F0-F1-F2) was studied, and the sensitivity of an organism to the action of carcinogen (Af mice) after a stay in the ChNPP exclusion zone was analyzed. It was ascertained that the most significant changes of the morphofunctional state of the animals were observed in the II generation (F2), which remained for a long period under the conditions of radioactive contamination. We have revealed an increased number of leukocytes, neutrophils, lymphocytes, and, especially, monocytes as against while the decrease in the number of erythrocytes and haemoglobin content; the decrease of thyroid function and cortical layer of the adrenals as opposed to while the increase in the relative weight of testes and their epididymides and the decrease in the number of spermatocytes and spermatozoa in the testis tissue. The exposure of Af mice in the exclusion zone increases the processes of mutagenesis and carcinogenesis, and changes the organism response to standardized action of chemical carcinogen. The increase in the exposure time of animals intensifies metabolic processes in a cell and increases their sensitivity to the action of xenobiotics.

A study assessing the genotoxicity in rats after chronic oral exposure to a low dose of depleted uranium.

PURPOSE: The aim of this study was to evaluate the potential genotoxicity induced by chronic oral exposure to depleted uranium (DU). MATERIALS AND METHODS: Weanling Wistar rats (F(0)), 50/sex/group, were exposed to DU in food at doses of 0, 4, or 40 mg kg(-1)day(-1) for four months. They were subsequently mated, resulting in the birth of F(1) rats. Fifty F(l) weanlings/sex/group were exposed for four months to the same dose levels as their parents. After four months, the uranium content in the tissues, the potential damage to the genetic material, and pathomorphological changes of the testicles were observed in both F(0) and F(1) rats. The genotoxicity of DU was evaluated by the following methods: sperm abnormality assessment, the bone-marrow micronucleus test, and the comet assay. RESULTS: Uranium content in F(1) rats was significantly higher than that in F(0) rats in both the kidney and ovary (p < 0.05). The sperm abnormality rate, marrow cell micronuclei rate, comet tail length, and tailed cell percentage increased in each treatment group in each generation compared with the control group (p < 0.05). When comparing F(1) with F(0) rats, significant differences were detected for most of the indicators, with F(1) rats always exhibiting more damage (p < 0.05). With regard to pathomorphological changes in the testicles, the sperm displayed atypical changes, including thickening of the anachromasis nucleolus, which seemed to be more severe in F(1) rats. CONCLUSION: Genotoxicity may be induced in rats after chronic oral exposure to a low dose of DU.

Protective effects of selenomethionine against ionizing radiation under the modulation of p53 tumor suppressor.

Ionizing radiation (IR) therapy has been widely employed in the treatment of cancer. However, certain issues, including toxicity, have been raised in conjunction with IR therapy for cancer. Recently, selenomethionine (SeMet) as an antioxidant has been the subject of a great deal of attention for its chemopreventive effects. In this study, we found that DNA repair activity has been enhanced in response to SeMet against IR. In addition, our data showed that p53 functional activity was significantly reduced against IR in the cells expressing a mutant form of redox factor 1 (Ref-1) contrast with Ref-1 wild-type cells treated with SeMet, suggesting that p53 activation under the modulation of Ref-1 might play an important role in IR-treated cells in the presence of SeMet. Furthermore, IR-induced micronuclei numbers were also reduced after treatment with SeMet, strongly implicating protection by SeMet in genomic stability against IR-induced genotoxicity. From this study, we suggest that the p53-mediated protective mechanism of SeMet might provide clues for reducing side effects of IR therapy.

Rosmarinic acid, a photo-protective agent against UV and other ionizing radiations.

Solar UV and other ionizing radiations cause a generation of reactive oxygen species, induce cellular DNA damage and alter skin homeostasis. The use of exogenous antioxidants is increasingly frequents, we attempt to demonstrate that a rosmarinic acid extract acts as photo-protector; both free radical scavenger as an inducer of the body's own endogenous defence mechanisms by regulating tyrosinase activity and stimulating melanin production. Malonyldialdehyde formation (TBARS) was delayed when RA was used. The protection factor was 3.24 times vs AA. TEAC value for RA was 1.6 times vs AA. The radioprotective-antimutagenic effects of RA were measure using the micronucleus test. The level of micronucleous for treatments before irradiation was: RA [14]

Effect of North American ginseng on 137Cs-induced micronuclei in human lymphocytes: a comparison with WR-1065.

To explore the radioprotective effect of a standardized North American ginseng extract (NAGE) on human peripheral blood lymphocytes (PBL), a micronuclei (MN) assay was conducted in PBL obtained from 12 volunteers. NAGE (50-1000 microg/mL) and WR-1065 (1 mM and 3 mM) were applied to PBL cultures at 0 h and 90 min post-irradiation. It was found that (1) the baseline MN yield of PBL ranged from 14.4 +/- 1.5 to 15.9 +/- 1.5 per 1000 binucleated cells (p > 0.05); after irradiation (1 Gy and 2 Gy), the MN yield increased sharply; (2) MN yields declined with increasing concentrations of NAGE and WR-1065. Even at 90 min post-irradiation of 1 Gy, the maximum level of MN reduction rate caused by NAGE and WR-1065 was 53.8% and 59.2%, respectively; after 2 Gy irradiation, it was 37.3% and 42%, respectively; (3) the MN distribution in PBL followed a non-Poisson distribution in all cases; and (4) both NAGE and WR-1065 showed no significant effect on the proliferation index of lymphocytes. The results indicate that NAGE is a relatively non-toxic natural product, which can be administered as a dietary supplement and has the potential to be a radiation countermeasure.

Protective effect of the cruciferous vegetable mustard leaf (Brassica campestris) against in vivo chromosomal damage and oxidative stress induced by gamma-radiation and genotoxic chemicals.

We evaluated the possible protective effect of the popular Indian cruciferous vegetable mustard leaf (Brassica campestris) against chromosomal damage and oxidative stress induced by gamma-radiation, cyclophosphamide (CPH) and urethane (URE), in mice. In vivo bone marrow micronucleus test was performed to assess chromosomal damage, and oxidative stress was monitored by estimating the changes in lipid peroxidation and the status of glutathione (GSH) as well as redox cycle antioxidants. Pretreatment with 50-250 mg/kg body wt of mustard leaf extract (MLE) for seven days significantly reduced the frequencies of micronuclei induced by gamma-radiation, CPH and URE. The protective effect against chromosomal damage was associated with modulation of lipid peroxidation as well as an increase in GSH and the GSH-dependent enzyme glutathione S-transferase (GST). Mass spectral analysis showed the presence of glucosinolates in MLE used for the pretreatment of mice. These findings indicate that intake of the green leafy cruciferous vegetable mustard leaf can lead to protection against in vivo genotoxicity and oxidative stress.

Radioprotective properties of the phytochemically characterized extracts of Crataegus monogyna, Cornus mas and Gentianella austriaca on human lymphocytes in vitro.

The objective of the present study is to evaluate the radioprotective properties of extracts of Crataegus monogyna Jacq. (Rosaceae) fruit, Cornus mas L. (Cornaceae) leaves and Gentianella austriaca (A. Kern. & Jos. Kern.) Holub (Gentianaceae) aerial parts on cultured human peripheral blood lymphocytes in vitro after irradiation with 2 Gy of 60Co gamma-rays. Plants were collected at Mt. Maljen in Serbia, air-dried and powdered, and the total phenolic content was analyzed. In C. mas leaves, ellagic and gallic acid were found to be the dominant compounds, whereas C. monogyna fruit was rich in procyanidins and flavonoids. The main constituents of G. austriaca aerial parts were gamma-pyrones and secoiridoids. Cell cultures were treated with four different doses of plant extracts (0.1 mg/mL to 0.4 mg/mL). Treatment with the lowest dose gave the best protective effect. Significant radiorecovery potentials of C. mas and C. monogyna were observed, seen as a reduced incidence of radiation-induced micronuclei (19.23% and 13.24%, respectively), reduced levels of lipid peroxidation products (50.04% and 13.18%, respectively) and two-fold enhanced apoptosis. Both extracts slowed down cell proliferation gradually, enabling more time for repair. G. austriaca possesses strong antioxidant properties, significantly reducing lipid peroxidation and the incidence of micronuclei (for 30.88% and 35.56%, respectively) while enhancing apoptosis with no perturbation of the cell cycle. This study may contribute to the search for novel radioprotective agents.

Kojic acid -absence of tumor-initiating activity in rat liver, and of carcinogenic and photo-genotoxic potential in mouse skin.

Kojic acid (KA) has been widely used as a quasi-drug ingredient. Possible promotion activity of KA was suggested on livers of mouse and rat by findings obtained in genotoxicity and carcinogenicity studies performed thus far. Therefore, in order to examine safety as a quasi-drug ingredient, we investigated the presence of initiation activity in rat liver and the photo-genotoxicity and carcinogenicity in mouse skin. In medium-term carcinogenesis test in rats, 2.0% KA was orally given to F344/DuCrj rats for 4 weeks of the initiation period, followed by the combination of partial hepatectomy and treatment with a hepatocarcinogenesis promoter, phenobarbital (PB). As a result, glutathione S-transferase placental form (GST-P) positive foci of 0.2 mm or more in diameter in the KA group, which is usually used in determination of pre-cancerous lesions, did not increase significantly in both numbers and areas compared with those of the non-initiated controls. In the concurrent analysis, however, numbers of GST-P-positive foci of two cells or more and 0.1 mm or more in diameter increased slightly, and possible weak initiation activity of KA was equivocal. However, considering the known fact that KA exerts promotion activity in the liver of F344 rats by long-term dietary administration, it was suggested that the observed slight increase of the numbers of GST-P-positive foci in rat liver was the effect of promotion activity of KA rather than the initiation activity. In DNA adducts formation assay in a rat liver, no clear adducts derived from KA were detected in male F344/DuCrj rats administered 0.5% or 2% KA orally, and KA was considered not to form DNA adducts in rat liver. In the in vitro photo-reverse mutation assay with bacteria, KA exerted weak photo-mutagenicity. Furthermore, in chromosome aberration study in Chinese hamster lung cells (CHL/IU cells) with UV irradiation, KA induced chromosome aberration at high-dose (1.4 mg/mL) treatment with UV irradiation, but was negative without UV irradiation. However, in the in vivo photo-micronucleus study in mouse, in which 1.0 or 3.0% KA containing cream was applied twice to the back of the animals with a 24-hr interval, KA did not induce micronuclei in mouse epidermal cells. Based on these results, it is considered that the risk of KA to exert photo-carcinogenicity is quite low in the skin. In skin carcinogenesis bioassay for initiation-promotion potential, 3.0% KA cream formulation was applied to the back of the mouse for 1 week (once a day, total 7 times) and for 19 weeks (5 times a week, total 95 times) during the initiation and the promotion stages, respectively. No skin nodules were observed in any animal skins formed due to KA treatment given in either stage. Therefore, KA is considered not to possess initiation nor promotion activity of skin carcinogenesis. Furthermore, from the above findings, it is suggested that KA is virtually safe as a quasi-drug ingredient.