IL-6 and IL-10 Levels in Rats Blood Plasma as Immune Responses Post Radioiodine (I-131) Administration.

OBJECTIVE: The purpose of this study was to analyze the effect of oral administration of radioiodine (I-131) on the immune responses (interleukin 6 and 10) as biodosimetry markers and to support clinical trials of I-131 solution. METHODS: The design of this study was an in vivo experimental study using twenty-seven male rats (Rattus norvegicus strain Sprague-Dawley) given 100 µL of I-131 solution at a dose of 260 µCi. Blood plasma was taken at 0.25, 0.5, 1, 3, 24, 48, 120, and 168 hours post oral I-131 administration, respectively. Rats without radioiodine administration as a control group. The levels of IL-6 and IL-10 were measured using the enzyme-linked immunosorbent assay (ELISA) method. Statistical analysis was carried out with one-way ANOVA using SPSS version 25 software. RESULT: IL-6 level began to significantly increase at 0.25 hours post administration of I-131 (14.4 pg/mL ± 2.52 pg/mL, p=0.02). During 7 days of observation, IL-6 levels had 2 peaks of highly significant increase at 0.5 hours (43.57 ± 5.28, p<0.001) and 120 hours (24.08 ± 2.69, p<0.001 compared to control (5.44 ± 0.95 pg/mL). IL-10 level began to significantly increase at 0.25 hours (30.32 ± 3.22 pg/mL, p=0.03) compared to controls (20.61 ± 1.59 pg/mL). CONCLUSION: The highest increase in IL-6 and IL-10 levels occurred respectively in the first 0.5 hours 8 times and in the first 0.25 hours 1.47 times compared to controls. Internal irradiation with radioiodine resulted in a significant increase in immune cells in exposed blood plasma characterized by the production of the cytokines IL-6 and IL-10. This appears to be a response of immune cells to reduce or stop inflammatory reactions through the release of anti-inflammatory cytokines in an effort to prevent excessive inflammatory responses that can damage cells and tissues.

The effect of red fruit oil (Pandanus conoideus Lamk.) emulgel on angiogenesis and collagen density in incisive wound healing in mice (Mus musculus).

Pandanus conoideus Lamk. or commonly known as red fruit oil (RFO) can be used to accelerate wound healing because it contains tocopherols, carotenoids, oleic acid, linoleic acid, and linolenic acid. The RFO in this study was formulated in the form of an emulgel because it has the most convenient and effective drug delivery system. The aims of this study were to determine the activity of RFO emulgel on increasing the amount of angiogenesis and collagen density in incised wound healing and to determine the optimal dose of RFO emulgel to increase the amount of angiogenesis and collagen density in incised wound healing. This was a true experimental study with a posttest only control group design that included five treatment groups: a positive control group (10% povidone-iodine), a negative control (gel base), and three groups that varied the concentration of RFO emulgel used at 5%, 10%, and 15%. Parameters observed were the amount of angiogenesis using Image Raster software and the percentage of areas of collagen density using ImageJ software. The data were analyzed using a one-way ANOVA test and continued with the least significant difference test. The results of this study showed that RFO emulgel was able to increase the amount of angiogenesis and collagen density in the wound healing process with P = 0.000. An increase in the amount of angiogenesis and collagen density occurred in mice treated with RFO compared to the positive and negative control groups. It can be concluded that RFO emulgel has activity toward increasing the amount of angiogenesis and collagen density in the wound healing of mice incisions. The optimal dose concentration of RFO emulgel for increasing the amount of angiogenesis and collagen density in incision wound healing was shown in RFO emulgel with a concentration of 15%.

Immune status of people living in the Tande-Tande sub-village (Indonesia), an area with high indoor radon concentration.

On Earth, there are significant variations in terms of exposure to naturally occurring radiation among different areas. Radon, a naturally-occurring radioactive gas that is the primary cause of lung cancer in nonsmokers and the second most prevalent cause among smokers, poses a considerable risk. Indoor radon, in particular, constitutes the most substantial source of natural radiation to which individuals are exposed. This study assessed the immune status of a population chronically exposed to high indoor radon concentration in Indonesia. Fifty-seven subjects from the Tande-Tande sub-village (high indoor radon concentration area) were compared to fifty-three participants living in the Topoyo village (low concentration area). We contrasted the immunological conditions of these two populations by measuring levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-4 (IL-4), and IL-10 in serum. Moreover, we also measured levels of the nuclear factor kappa B (NF-κB), superoxide dismutase (SOD), glutathione peroxidase (GPX), and protein kinase B in its phosphorylated (pAkt) and non-phosphorylated form (Akt) in peripheral blood mononuclear cells (PBMCs) of a subset of participants (31 from each population). TNF-α, IFN-γ, and IL-4 levels in Tande-Tande sub-village inhabitants were significantly lower than those in the control group living in the Topoyo village (p = 0.001, p = 0.017, and p = 0.002). The concentration of IL-10 also tended to be lower in people living in the high indoor radon concentration area, but it did not differ significantly between Tande-Tande sub-village inhabitants and Topoyo inhabitants (p = 0.106). Protein levels of NF-κB, pAkt, and Akt in Tande-Tande sub-village inhabitants also did not differ significantly between Tande-Tande sub-village inhabitants and Topoyo inhabitants (p = 0.234, p = 0.210, and p = 0.657). Similarly, activities of SOD and GPX did not differ significantly between the two populations (p = 0.569 and p = 0.949). Overall, despite their chronic exposure to high indoor radon concentrations, our study revealed no increase in the levels of TNF-α, IFN-γ, IL-10, IL-4, SOD, and GPX in the inhabitants of Tande-Tande sub-village compared with people living in the Topoyo village. Furthermore, our study demonstrated no activation in the Akt pathway, as indicated by the pAkt/Akt ratio observed in PBMC lysates of individuals residing in the Tande-Tande sub-village.

γ-H2AX and phospho-ATM enzyme-linked immunosorbent assays as biodosimetry methods for radiation exposure assessment: a pilot study.

In the event of a radiological incident, a fast and accurate biological dosimetry (biodosimetry) method for evaluating people who have been potentially exposed to ionising radiation is crucial. Among the many biodosimetry methods available, the immunodetection of phosphorylated H2AX (γ-H2AX) stands as a promising method to be used in the triage of patients exposed to radiation. Currently, the most common way to measure γ-H2AX levels is through fluorescence microscopy. In this pilot study, we assessed the feasibility of using an enzyme-linked immunosorbent assay (ELISA) for quantifying γ-H2AX for biodosimetry purposes. Moreover, the usefulness of measuring phosphorylated ATM (pATM) levels through ELISA for biodosimetry was also evaluated. Blood samples were obtained from three male donors (38 y) and were irradiated with 60Co (0, 1, 2 and 6 Gy). Peripheral blood mononuclear cells (PBMCs) were isolated and lysed before measuring γ-H2AX, total H2AX protein and pATM using ELISA kits. The dicentric chromosome assay (DCA) using whole blood was also performed for comparison. Data from all donors at each dose were pooled before statistical analysis. The ratio of γ-H2AX/total H2AX and pATM levels increased in a radiation-dose-dependent manner. The average γ-H2AX/total H2AX ratios were 0.816 ± 0.219, 0.830 ± 0.685, 1.276 ± 1.151 and 1.606 ± 1.098, whereas the average levels of pATM were 59.359 ± 3.740, 63.366 ± 0.840, 66.273 ± 2.603 and 69.936 ± 4.439, in PBMCs exposed to 0, 1, 2 and 6 Gy, respectively. The linear-quadratic dose-response calibration curve for DCA was Y = 0.0017 (±0.0010) + 0.0251 (±0.0142) × D + 0.0342 (±0.0039) × D2  $\boldsymbol{Y}=\mathbf{0.0017}\left(\pm \mathbf{0.0010}\right)+\mathbf{0.0208}\left(\pm \mathbf{0.0218}\right)\times \boldsymbol{D}+\mathbf{0.0350}\left(\pm \mathbf{0.0050}\right)\times{\boldsymbol{D}}^{\mathbf{2}}$. Overall, despite a large variability in the ratio of γ-H2AX/total H2AX among donors, the present study revealed the suitability of using the ratio of γ-H2AX/total H2AX and pATM for biodosimetry. Still, more research with a larger group of subjects is necessary to construct a reliable calibration curve for the ratio of γ-H2AX/total H2AX and pATM levels for biodosimetry.

Preliminary study of chromosome aberrations using Giemsa, two-colour fish, and micronucleus assays in lymphocytes of individuals living in elevated radon concentration areas.

The frequencies of unstable and stable chromosome aberrations and micronuclei were examined in peripheral blood samples from 10 individuals living in elevated radon concentration areas (Takandeang Village, Mamuju, Indonesia). Blood samples from 10 people living in Topoyo Village were used as a control group. For unstable chromosome aberration analysis, a dicentric chromosome assay was conducted using conventional Giemsa staining. Chromosomal painting of chromosomes 1 and 4 using the fluorescence in situ hybridisation technique was also applied to four subjects to assess the stable chromosome aberration. Our study showed no significant increases across all groups in dicentric and other unstable chromosome aberrations, such as rings and acentric fragments. Translocations were found in one person from Takandeang Village and two Topoyo Village inhabitants. The translocations found in the subjects from Takandeang Village were due more to aging factors than to radon exposure. The number of micronuclei per 1000 binucleus cells in Takandeang Village inhabitants was not significantly different than that in the control group (p = 0.943). A more comprehensive analysis should be conducted in a subsequent study by increasing the number of study donors and the number of metaphases to be analysed in both dicentric chromosome assay and fluorescence in situ hybridisation assays. Such research could provide valid information on the cytogenetic effects of elevated indoor radon exposure.

Chromosome aberrations, micronucleus frequency, and catalase concentration in a population chronically exposed to high levels of radon.

PURPOSE: To deepen our knowledge on the effects of high levels of indoor radon exposure, we assessed the frequencies of unstable and stable chromosome aberrations and micronucleus (MN), as well as the concentration of an endogenous antioxidant (catalase, CAT), in blood samples of individuals chronically exposed to high indoor radon concentrations in Indonesia (Tande-Tande sub-village, Mamuju, West Sulawesi). Moreover, we also investigated the occurrence of a radio-adaptive response (RAR) in Tande-Tande sub-village inhabitants using the G2 MN assay. MATERIALS AND METHODS: The frequencies of dicentric (DC), acentric (AF), ring (R), and translocation (Tr) chromosomes in Tande-Tande inhabitants were compared to those in people living in a reference area with low levels of indoor radon levels (Topoyo village, Indonesia). The number of MN per 1000 binucleated cells (BNC) and CAT concentration per total protein was quantified and compared between groups. Lastly, we irradiated (2 Gy) phytohemagglutinin-stimulated samples in vitro and measured the frequency of MN to verify the occurrence of a RAR in Tande-Tande sub-village inhabitants. RESULTS AND CONCLUSION: The frequencies of DC, AF, and Tr did not differ between Tande-Tande inhabitants and control subjects (p = 0.350, 0.521, 0.597). The frequency of MN in Tande-Tande inhabitants was significantly lower than that in the control group (p = 0.006). Similarly, CAT concentration in Tande-Tande inhabitants was also significantly lower than that in the control population (p < 0.001). Significant negative correlations were identified for MN number and CAT concentration versus indoor radon concentration, annual effective dose, or cumulative dose both within groups and when all data were analyzed together. Our findings indicate that, despite the high indoor radon levels, Tande-Tande inhabitants are not under oxidative stress, since this group had lower CAT concentration and MN frequency than those in the control group. The negative correlation between MN frequency and indoor radon concentration, annual effective dose, and cumulative dose suggests the occurrence of an RAR phenomenon in Tande-Tande sub-village inhabitants. This interpretation is also supported by the results of the G2 MN assay, which revealed lower MN frequencies after in vitro irradiation of samples from Tande-Tande sub-village inhabitants than those in samples from the control group (p = 0.0069, for cumulative MN frequency; p = 0.0146, for radiation-induced MN only).

Detection of biological responses to low-dose radiation in humans.

It has been considered difficult to detect the biological effects of low-dose radiation exposure below approximately 100 mSv in humans. Serum proteomic analysis and oxidative modification profiling were conducted with blood samples collected from residents of a newly discovered high-level natural background radiation area (annual effective dose approximately 50 mSv y-1) and normal-level area (1.22 mSv y-1) in Mamuju, Indonesia, where many people have been living for generations. Dose-dependent oxidative modifications in amino acid sequences of human serum albumin, especially the 162nd and 356th tyrosine residues and 111th and 470th methionine residues, were found. None of these findings have been reported in humans exposed to chronic low-dose radiation. It can be used as a biomarker not only for the assessment of the presence or absence of radiation exposure but also for dose prediction in living organisms for chronic radiation. These results suggest that traces of radiation exposure are recorded in serum albumin and that there is a possibility of a new methodology that can evaluate biological responses below 100 mSv.

Superoxide dismutase and glutathione peroxidase activities in a population exposed to high indoor radon concentration: a preliminary report.

The objective of this study was to assess the effect of radon exposure on the modulation of endogenous antioxidants in a population chronically exposed to high levels of radon indoors. To do so, we measured the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) in peripheral blood mononuclear cells (PBMCs) of people living in an area with high indoor radon concentration (Tande-Tande sub-village, Indonesia). The activities of SOD and GPX in Tande-Tande inhabitants were compared with those in subjects living in the Topoyo village (Indonesia), an area with low indoor radon levels. The activities of SOD and GPX in Tande-Tande sub-village inhabitants did not differ from those in people living in the Topoyo village (0.37 ± 0.021 versus 0.28 ± 0.018 U/mg protein and 8.46 ± 1.48 versus 8.34 ± 1.65 U/mg protein, p > .05). For both populations, there was a significant positive correlation between SOD and GPX activities (p < .001). No significant effects of gender, age, smoking habit, and body mass index on SOD and GPX activities were found for both groups. Although no significant modulation of SOD and GPX activities in PBMCs was detected, further studies should expand the sample size and also assess antioxidant levels in the serum. This study provides a first picture of endogenous antioxidant systems in Tande-Tande sub-village inhabitants, but a more comprehensive analysis, including the measurement of catalase (CAT) activity, might provide additional insight into the effects of chronic exposure to high indoor radon concentrations.

Prediction of compounds with antiosteoporosis activity in Chrysophyllum cainito L. leaves through in silico approach.

OBJECTIVES: Estrogen deficiency causes various health problems in postmenopausal women, including osteoporosis. Phytoestrogen emerged as a potential alternative of estrogen with minimum side effects. The aims of this study were to analyze the metabolite profiling results of various extract of Chyrsophyllum cainito L. leaves, which contain phytoestrogen, through in silico study against 3OLS protein, an X-ray protein of ERß, so it can predict the types of the phytoestrogen contents which have antiosteoporosis property. METHODS: In silico analysis was carried out for the compounds from the metabolite profiling data of C. cainito leaves from our previous study. The structure compounds from metabolite profiling results of various extract of C. cainito leaves were prepared with Avogadro 1.0.1 software, molecular docking was done using PyRx 0.8 software, and Biovia Discovery Studio Visualizer 2016 software was used to visualize the structure of compounds against 3OLS protein. The physicochemical characteristics of the compounds were analyzed using the SwissADME web tool. RESULTS: From in silico studies, it was known that there were total 11 compounds in C. cainito leaves that predicted as phytoestrogens which have ERß agonist properties against 3OLS protein. The ERß agonist was a compound that has parameters similar to 17ß-estradiol in its interaction with 3OLS protein, which has a pharmacophore distance of 10.862 Å, and binding to amino acids His 475 and Glu 305 or Arg 346 at receptor-ligand docking simulation. CONCLUSIONS: C. cainito leaves contain 11 compounds that are predicted to be phytoestrogens with ERß agonist properties, which is responsible for antiosteoporosis activity.

Evaluation of sodium orthovanadate as a radioprotective agent under total-body irradiation and partial-body irradiation conditions in mice.

PURPOSE: Our previous study indicated that sodium orthovanadate (vanadate), a strong inhibitor of p53, effectively suppressed the lethality from the hematopoietic (HP) and gastrointestinal (GI) syndromes after 12 Gy total-body irradiation (TBI) in mice. This conclusion, however, was inconsistent with the fact that p53 plays a radioprotective role in the intestinal epithelium. The death after TBI of around 12 Gy was attributed to a combined effect of HP and GI syndromes. To verify the effect from prophylactic administration of p53 inhibitor on protection of HP and GI syndromes, in this study, the radioprotective effects from vanadate were investigated in TBI and lower half-body irradiation (partial-body irradiation: PBI) mouse models. METHODS: Female ICR mice were given a single injection of vanadate or vehicle, followed by a lethal dose of TBI or PBI. Radioprotective effects of vanadate against the irradiations were evaluated by analyzing survival rate, body weight, hematopoietic parameters, and histological changes in the bone marrow and intestinal epithelium. RESULTS: TBI-induced HP syndrome was effectively suppressed by vanadate treatment. After TBI, the vanadate-treated mice retained better bone marrow cellularity and showed markedly higher survival rate compared to the vehicle-treated animals. In contrast, vanadate did not relieve loss of intestinal crypts and failed to rescue mice from GI death after PBI. CONCLUSION: Vanadate is a p53 inhibitor that has been shown to be beneficial as a radiation protective agent against HP but was not effective in protecting against acute GI radiation injury.

Development of Dose-Response Calibration Curve for Dicentric Chromosome Induced by X-Rays.

Chromosome aberration is a biomarker that has been used as a standard tool in biological dosimetry (biodosimetry) of individuals after exposure to ionizing radiation. It is based mainly on the induction of dicentric chromosomes - one of the radiation-induced biological effects, in order to correlate them with radiation dose. In this study, a dose calibration curve for X-rays was generated by using the dicentric assay and by fitting the data to both Chromosomal Aberration Calculation Software and Dose Estimate programs to compare the output of each method. Peripheral blood samples from four nonsmoker healthy donors were irradiated with various doses ranging from 0 to 4 Gy with 250 kV or 122 keV X-rays at a dose rate of 0.17 Gy/min. The irradiated blood was cultured, harvested, and analyzed according to the standard procedure as described by the International Atomic Energy Agency with slight modifications. The dose-response calibration data for dicentrics were fitted with the linear-quadratic model (Ydic = 0.03987D2 + 0.00651D). The dose-response calibration curve obtained in this research was comparable to other estimations with similar radiation quality and dose rates. The results in this research convinced us in sustaining a biodosimetry using a dose-response calibration curve in our laboratory.

Study of γ-H2AX as DNA double strand break biomarker in resident living in high natural radiation area of Mamuju, West Sulawesi.

High expression of phospho histone γ-H2AX, a sensitive marker of double stranded DNA damage, is believed to be an indication of defective DNA repair pathway or genomic instability that may cause mutations and ultimately cancer. DNA damage can be caused by ionizing radiation exposure. Beside in medical treatment/diagnosis or industry, ionizing radiation exposure can also be found in naturally in regions of high natural back ground radiation. In this study we collect the blood from 45 volunteers living in Mamuju, a region with highest natural radiation in Indonesia (dose of ∼7 mSv/year). Subjects were grouped as high natural background area (HNBA) (n = 37) and control area (n = 8). The expression γ-H2AX foci were evaluated by one of researcher fluorescence microscope examination. Our results show that the average foci numbers per cell were in the normal range. While not statistical different, the average of γ-H2AX foci in exposed area higher in the exposed compared to the control area, 0.31 versus 0.13 (p > 0.05), respectively. Moreover, there was also no statistical difference of average γ-H2AX foci between man and woman, old and young people in exposed and control area (p > 0.05). In this preliminary study we find that γ-H2AX foci (and thus DNA double strand break) frequency in residents living in the HNBA of Mamuju, West Sulawesi, show a trend towards higher (albeit not significant) average values relative to the control area. More research is needed to further scrutinize these observations.