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The aim of this study is to explore the genotoxicity of cells obtained from the buccal mucosa in patients who were exposed to dental X-rays using micronucleus analysis. All the subjects underwent a routine oral clinical examination and subjects with any visible or symptomatic change in the buccal mucosa were excluded. Subjects who were expose to X rays in the past 6 months were also excluded. Based on the inclusion and exclusion criteria a total of 116 subjects were recruited. The included subjects were all nonsmokers. The genotoxicity was studied by micronucleus assay. There was significant difference in the frequency of multinucleated cell numbers from before exposure to after exposure to OPG. In patients having exposed to CBCT, a higher cell turnover was detected. The number of multinucleated cells gradually increases after panoramic radiographs, hence dental X-rays should be prescribed only when absolutely necessary.

Oral squamous cell carcinoma (OSCC) is the most commom cancer in the world. If remain untreated for several years, it may be fatal. Hence, it is important to prevent and treat OSCC at an early stage. In this study the effect of aqueous and dry leaves extract of Ocimum sanctum was observed on Ca9-22 cell line, which is an OSCC cell line. For this, Ca9-22 cell line was cultured and maintained. After 24 h, the cells were treated with aqueous and dry leaves extract of Ocimum sanctum plant. Viability of the cancerous cells were studied by 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and neutral red uptake (NRU) assay. Minimum inhibitory concentrations (MIC), lethal concentration25 (LC25), lethal concentration50 (LC50) and highest permissive concentration (HPC) was calculated by probit computational method. Experimentally, the MIC value was 5 mg/L, whereas the HPC was 30 mg/L of the plant extract in aqueous state. For the dry extract the MIC was 5 mg/L whereas the HPC was 35 mg/L for both MTT and NRU assays. For MTT assay LC values: 7.41 (LC25), 14.79 (LC50) and 26.91 mg/L (LC75) for aqueous extract and 12.58 (LC25), 20.89 (LC50), 29.51 mg/L (LC75) for dry extract. For NRU assay LC values were 10.23 (LC25), 14.79 (LC50) and 20.89 mg/L (LC75) aqueous extract, and 16.59 (LC25), 23.44 (LC50), 30.19 mg/L (LC75) dry extract of the plant. From the above study it was concluded that, Ocimum sanctum have anti-cancerous activity. It can further be used for therapeutic purposes.

Background Leukoplakia generally refers to a firmly attached white patch on a mucous membrane which is associated with an increased risk of cancer. So it is important to diagnose leukoplakia in early stage. In this study we are trying to treat leukoplakia as a marker for prediagnosis of cancer. Since, bleomycin as a chemotherapeutic agent, is mainly used in the treatment of multiple tumors, and several cancers. Hence in this study effect of bleomycin was studied in D38, a leukoplakia cell line.Methods Percent lethality values of leukoplakia cells after treated with several concentations of bleomycin was studied by acridine orange/ethidium bromide staining (AO/EB), propidium iodide (PI) staining, 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and comet assay. Minimum inhibitory concentration (MIC), lethal concentration25 (LC25), lethal concentration50 (LC50), lethal concentration75 (LC75) and highest permissive concentration (HPC) was calculated from probit computational method.Results From AO/EB staining, PI staining, MTT assay and comet assay the MIC value was 15 mg/L concentration of bleomycin, where as the HPC was found at 100 mg/L of bleomycin. The comet tail length gradually increased from 15 mg/L to 100 mg/L of bleomycin. Percent lethality values were also increased from 15 to 100 mg/L concentration of bleomycin in all the staining protocols as well as in comet assay technique.Conclusion It is important to diagnose leukoplakia in early stage. Since, bleomycin as a chemotherapeutic agent, it is mainly used in the treatment of multiple tumors, testicular carcinomas, lymphomas, and head, neck cancers. From the results, it was found that bleomycin can be used for treatment of oral leukoplakia to prevent its development to SCC.

Toxicities of methylmercury chloride (CH3HgCl) and methylmercury hydroxide (CH3HgOH) to cultured neuroblastoma cell line SH-SY5Y in vitro are evaluated. This is the comparative study between two methylmercury compounds to find out the extent of toxicity of these compounds are toxic to SH-SY5Y cell line. Both cytotoxicity and genotoxicity experiments were carried out to find out the more toxic compound. For cytotoxicity study, four staining assay methods independently with trypan blue (TB), acridine orange/ethidium bromide (AO/EB), 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT), and neutral red (NR) were used and the comet assay method was done for genotoxicity study. The obtained toxicity data were used for probit analysis. In cytotoxicity, CH3HgCl had minimum inhibitory concentration (MIC) value in each assay method as 3 mg/L invariably; LC25 values were in the range 7.41 to 10.23 mg/L, and LC50 values were 14.79 to 15.48 mg/L; while LC75 values were 20.89 to 26.91 mg/L. Moreover, LC100 value was 30 mg/L, known from comet assay experiments for CH3HgCl. Similarly for CH3HgOH, the MIC value in each assay method was invariably 3 mg/L, the LC25 values were in the range 12.58 to 16.59 mg/L, and LC50 values were 19.49 to 23.44 mg/L; LC75 values were 27.54 to 30.90 mg/L and LC100 value was 42 mg/L in each assay done for cytotoxicity and genotoxicity studies. Computed DNA fragmentation indices in comet assays were 98.6 ± 0.57 30 mg/L with CH3HgCl and 76 ± 5.29 30 mg/L with CH3HgOH. This study clearly indicated that methylmercury chloride is more toxic than methylmercury hydroxide to SH-SY5Y cell line. Toxicity of Hg had been quantified with in vitro cultured human neuroblastoma cell line; since it has neurotoxic effects, its neural evaluation has implications in environmental health issues.

Human dental pulp stem cells (hDPSCs) are promising source of cells for numerous and varied regenerative medicine applications as those possess high proliferation potential with multilineage differentiation capacity compare to other sources of adult stem cells; therefore, hDPSCs could be the good source for autologous transplantation in tissue engineering and regenerative medicine. In this study stem cells were isolated from dental pulp and were characterised by flowcytometry and immunocytochemistry. The controlled cells as well as, 7-day cultured cells were positive for transcription factors, OCT 4 and SOX 2 thatconfirmed isolated cellsasmesenchymal stem cells (MSCs). These cells showed positive expression for CD 19, CD 73, CD 90, CD 105 and are negative for CD 34, CD 45. Viability of hDPSCS were studied by trypan blue (TB) staining and fluorescent microscopic study. After 7 days of passaging by using several growth factors, cells express neural cell markers oligodendrocyte and glial fibrillary acidic protein. Specifically, osteocytes were grown from dental pulp MSCSsin vitro with the help of growth factors, dexamethasone, ascorbic acid-2- phosphate and β-glycerophosphate whereas, adipocytes were grown with indomethacin, 3-isobutyl-1-methylxanthine and insulin. Osteocytes and adipocytes were characterized by von Kossa and Oil red O staining, respectively. Chromosomal analysis of dental pulp-MSCs was done for qualitative assessment of MSCs. Karyotyping indicated diploid chromosome number in dental pulp derived MSCs. In vitro grown osteocytes could be used for bone fracture reunion cases, and adipocytes could be used for further research purposes., Biological sciences; Cell biology; Stem cells research; Biotechnology; Health sciences; Dentistry; Dental pulp; Stem cells; Neural cells; Flow cytometry; Immunocytochemistry.

The present study examines the toxicity of HgCl2, using in vitro grown lymphocytes from human umbilical cord blood. Lymphocytes were diluted with Dulbecco’s modified Eagle’s medium, and were grown in the presence of grading doses of HgCl2. The cell-toxicity on grown lymphocytes was assessed using three staining procedures, trypan blue (TB) assay, acridine orange/ethidium bromide (AO/EB) staining and propidium iodide (PI) staining cytologically; and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. The standard procedure was carried out for monitoring the live cell density. Additionally, the comet assay was done to ascertain levels of HgCl2 in inducing the nuclear disruption of lymphocytes. Mean results of three independent experiments are presented. Probit-computed lethal concentration (LC) values obtained were in ranges, 5.00–16.22 mg/l as LC25, 13.18–24.54 mg/l as LC50, and 25.12–34.67 mg/l HgCl2 as LC75, respectively at 24 h with incubation with HgCl2, using TB, AO/EB, PI staining procedures and the MTT assay. Experimentally, the minimum inhibitory concentration (MIC), the highest permissive concentration (HPC) and the LC100 values were in ranges, 5, 35–40 and 40–45 mg/l, respectively, for cytotoxicity. With comet assay data, the MIC value was 5 mg/l, whereas the HPC and LC100 values were 35 and 40 mg/l, respectively; and LC25, LC50 and LC75 values were 15.48, 20.41 and 28.84 mg/l HgCl2, respectively. Thus, HgCl2 levels higher than 5 mg/l and below 40–45 mg/l were toxic to in vitro cultured human lymphocytes.

This work describes that cytotoxicity of lead chloride and lead acetate to in vitro cultured lymphocytes from human umbilical cord blood, using four monitoring methods namely, trypan blue staining, acridine orange/ethidium bromide staining, 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide (MTT) and neutral red uptake assays; lead genotoxicity to lymphocytes was monitored by comet assay. The MIC value in each method was invariably 300 mg/L for PbCl2. Lethal concentration25 (LC25) values were almost in an agreeable range: 691.83 to 831.76 mg/L; LC50 values in each method were almost in the range: 1174.9 to 1348.9 mg/L; LC100 values were in the range: 3000 to 3300 mg/L, for lead chloride. Similarly, The MIC value in each method were invariably 150 mg/L; LC25 values were almost in the range: 295.12 to 371.53 mg/L; LC50 values were in the range: 501.18 to 588.84 mg/L; LC100 value was 1500 mg/L in all assays, for lead acetate. The comet assay also indicated that the LC100 values were 3300 mg/L lead chloride and 1500 mg/L lead acetate. Thus, both cytotoxicity and genotoxicity were recorded at 3300 mg/L lead chloride and 1500 mg/L lead acetate with lymphocytes.

Toxicities of methylmercury chloride (CH

The aim was to investigate whether a human neural stem cell (NSC) line derived from human umbilical cord blood (hUCB) can be used for toxicity study. Toxicity of both neurotoxic environmental xenobiotics, methyl mercury chloride (CH3HgCl), lead acetate (CH3COOPb), and chlorpyrifos (CP), and non-neurotoxic insecticide, dichlorvos, as well as non-neurotoxic drugs, theophylline and acetaminophen were assessed. Additionally, differentiation of neuronal and glial cell lines derived from hUCB was elucidated. It was observed that CH3HgCl was more toxic to human NSCs in comparison to CH3COOPb and CP. The minimum inhibitory concentration (MIC) value against NSCs was 3, 10, and 300 mg/L, in each staining process, acridine orange/ethidium bromide (AO/EB) staining, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay, and Hoechst staining, for CH3HgCl, CP, and CH3COOPb, respectively. CH3HgCl had the LC25 value as 10.0, 14.4, and 12.7 mg/L, by staining method mentioned in succession. CP had the LC25 value as 21.9, 23.7, and 18.4 mg/L; similarly, CH3COOPb had LC25 values, successively as 616.9, 719.2, and 890.3 mg/L. LC50 values ranged from 18.2 to 21.7 mg/L for CH3HgCl, 56.4 to 60.2 mg/L for CP, and 1000 to 1460.1 for CH3COOPb. Theophylline, acetaminophen, and dichlorvos had no impact on the viability of NSCs. This work justified that hUCB-NSC model can be used for toxicity study.

Insecticides and their residues are known to cause several types of ailments in human body. An attempt had been made to assess digitally the geno-toxicity of methyl parathion (MP) and chlorpyrifos (CP) to in vitro-grown HepG2 cell line, with Hoechst 33342 staining, comet, and micronucleus assays. Additionally, "acridine orange/ethidium bromide" (AO/EB) staining was done for the determination of insecticide-induced cytotoxicity, in corollary. Hoechst 33342 staining of cells revealed a decrease in live cell counts at 8-40 mg/L MP and 15-70 mg/L CP. Moreover, nuclear fragmentations in ranges 8 to 40 mg/L MP and 15 to 70 mg/L CP were recorded dependant on individual doses, increasingly with concomitant increases in comet tail length values. DNA fragmentation index measured in comet assays was 94.3 ± 0.57 at 40 mg/L MP and 93.3 ± 2.08 at 70 mg/L CP. Average micronuclei number was 59.0 ± 2.00 at 40 mg/L MP and 62.6 ± 1.52 at 70 mg/L CP, per 1000 cell nuclei, in micronucleus assay. Minimum inhibitory concentration (MIC) values with AO/EB staining for monitoring cytotoxicity were 4 and 10 mg/L for MP and CP, respectively. Lethal concentration50 (LC50) values were 20.89 mg/L MP and 79.43 mg/L CP in AO/EB staining, for cytotoxicity with probit analyses. It was concluded that MP was comparatively more geno-toxic than CP to HepG2 cell. It was discernible that at lower levels of each insecticide, geno-toxicity was recorded in comparison to cytotoxicity.

Background/Aims To study the in vitro antibacterial activity of methanolic leaf extract of Combretum albidum in combination with the antibiotic ceftriaxone (CE) against multidrug-resistant (MDR) Pseudomonas aeruginosa, and to assess the host toxicity of the leaf extract with human cord blood-derived lymphocytes in vitro. Methods The synergistic/antagonistic action of the leaf extract with CE was evaluated with the checkerboard procedure. The leaf extract and the antibiotic were added into the wells of a microtiter plate in serially proportionate combinations along with nutrient broth, bacterial inocula, and 2,3,5-triphenyltetrazolium chloride to achieve visible growth of the bacterium. Lymphocytes from human cord blood were cultured and the toxicity of the leaf extract was assessed by both acridine orange/ethidium bromide (AO/EB) staining method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The test results were analyzed by the probit method. Results Minimum inhibitory concentration (MIC) values of the methanolic leaf extract and CE separately were 0.866 and 0.0384 mg/mL, respectively. The MIC value of the leaf extract with the antibiotic was in the range of 0.102–0.866 mg/mL. Synergistic results in the in vitro control were obtained in all combinations of the antibiotic and the extract (i.e., from 9:1 to 5:5); ratios 1:9, 2:8, and 3:7 caused antagonism, whereas an indifference was seen at the ratio 4:6. Individual minimum bactericidal concentration (MBC) values of the leaf extract and CE were 4.39 and 87.7 μg/mL, respectively, whereas those of all combinations, 9:1 to 1:9, ranged from 0.33 to 3.07 mg/mL. The MBC value of the leaf extract alone was 4.39 mg/mL, whereas in various combinations with CE (at ratios from 9:1 to 1:9), the value varied from 0.70 to 3.07 mg/mL. The MBC value of CE was only 0.877 mg/mL in all combinations with the leaf extract. The plant extract level of 58.88 mg/mL was considered the lethal concentration 25 (LC25) because of its lethal effects on lymphocytes, whereas the LC25 value for CE was 380.19 μg/mL according to the AO/EB staining method. In the MTT assay, however, the LC25 value of leaf extract was 53.70 mg/mL. Therefore, we used a leaf extract concentration of 50 mg/mL, which was a little below the LC25 value of leaf extract obtained in the MTT assay (i.e., 53.70 mg/mL). Conclusion This study provided the result of synergistic activity of the combination of crude leaf extract of C. albidum with CE against MDR P. aeruginosa. The leaf extract was nontoxic to human lymphocytes.

ObjectivesTo locate a plant with suitable phytochemicals for use as antimicrobial agents to control multidrug-resistant (MDR) bacteria as a complementary medicine, without host toxicity as monitored through cultured lymphocytes from human umbilical cord blood.MethodsThe methanol crude leaf extract of the plant Woodfordia fruticosa was subjected to antimicrobial assay in vitro with nine pathogenic MDR bacteria from clinical samples. This was followed by bioassay-guided fractionation with seven non-polar to polar solvents, gas chromatography–mass spectrometry analysis of the n-butanol fraction, and monitoring of the host toxicity of the leaf extract with in vitro grown lymphocytes from human umbilical cord blood.ResultsThe leaf extract of W. fruticosa had a controlling capacity for MDR bacteria. The minimum inhibitory concentration and minimum bactericidal concentration of the n-butanol fraction were