Evaluation of the probable synergistic toxicity of selected potentiated antiretroviral and antibiotics on some aquatic biomarker organisms.

Environmental effects of active pharmaceutical compounds (APCs) in the environment are not well characterized, hence the need for comprehensive evaluation. This study employed three bioassays using three organisms, namely, Allium cepa, Daphnia magna, and Salmonella typhimurium, in the ecotoxicity study of lone and a mixture of selected APCs, namely, lamivudine (L), an antiretroviral, and ciprofloxacin (C) and sulfamethoxazole (S), antibiotics, at a concentration range between 10 and 100 ppb, in order to evaluate the potential of the lone and ternary mixture to exert synergistic toxicity. Study results from exposure to lone APCs showed that the L, C, and S trio individually had fatal impacts on daphnids, with mortality rates of 100, 75, and 95%, respectively, after 48 h. Sulfamethoxazole showed a mutagenic tendency, with a mutation ratio (background/sample ratio) of 2.0. Lamivudine showed a lethal impact on the root length of A. cepa (p > 0.05, p = 3.60E-3). Further microscopic examination of the A. cepa root tip revealed chromosomal aberrations on exposure to each compound. The LCS-mix ecotoxicology bioassays indicated a synergistic effect on the daphnids, probably due to potentiation. Although the LCS mix had a cytotoxic effect (evidenced by the absence of bacteria colonies) on exposed TA 98 P450 Salmonella typhimurium strain, this effect was not observed in other bacterial strains. Microscopic examination of A. cepa exposed to the LCS-mix revealed an aberration in the mitotic stage of the cell. The impact of combination of the pharmaceuticals in aqueous ecosystems was greater than when exposed to the tested individual pharmaceutical compounds. Study result showed that these compounds have tendencies to pose a higher risk to exposed living entities when in combined/potentiated forms, and this could lead to distortion of the regular functioning of the ecosystem, particularly bacterial and other microbial populations that are listed among primary producers of the aquatic food web.

Analysis of high risk factors for chromosomal aberrations in peripheral blood lymphocytes of 200 radiation workers in Hainan Province / 上海预防医学

ObjectiveTo investigate the current status and high-risk factors of chromosomal aberrations in peripheral blood lymphocytes （PBL） of radiation workers in Hainan Province. MethodsA total of 200 radiological workers who underwent occupational health examination in Hainan Provincial Hospital of Traditional Chinese Medicine from January 2021 to December 2021 were selected to collect the occupational health examination data and the rate of PBL chromosomal aberrations. The influencing factors of PBL chromosomal aberrations were analyzed by logistic regression model. The predictive value of logistic regression prediction model on PBL chromosomal aberrations were determined by using the reciver operator characteristic （ROC） curve. ResultsA total of 20 000 cells （100 cells/person） were tested. The chromosomal aberration rate was 0.37% （74/20 000） and the PBL chromosomal aberration rate in the subjects was 6.00% （12/200）. Univariate analysis showed that PBL chromosomal aberrations in radiological workers were related to age， length of service， type of work and education （all P<0.05）， but not to gender （P>0.05）. The logistic regression prediction model was constructed based on the influencing factors， with χ2=9.413， df=9， P=0.852， suggesting a good model fit. The logistic regression prediction model predicted the area under the curve （AUC） for the occurrence of PBL chromosomal aberrations in radiation workers was 0.914 （95%CI： 0.866‒0.949）， with a cut-off value of 3.05， corresponding to a prediction sensitivity and specificity of 100.00% and 75.98%， respectively. ConclusionThe incidence of PBL chromosomal aberrations in radiological workers in Hainan Province was 6.00%， with age， working age and job type as high-risk factors and education level as a protective factor. The prediction model constructed by the above factors can provide a reliable basis for clinical prediction of PBL chromosomal aberrations in radiological workers.

Evaluation of the cytotoxic, anticancer, and genotoxic activities of Acacia nilotica flowers and their effects on N-methyl-N-nitrosourea-induced genotoxicity in mice.

PURPOSE: In this study, two main research objectives were examined: (1) the cytotoxic and anticancer activities of the aqueous methanol extract from Acacia nilotica flowers on three human cancer cells, namely lung A549, breast MCF-7, and leukemia THP-1 cells, and (2) the genotoxic effects of A. nilotica extract and its influence on DNA damage induced by N-methyl-N-nitrosourea (MNU) in mice. METHODS: Mice were orally treated with A. nilotica extract (200, 500, and 800 mg/kg for 4 days) with or without MNU (80 mg/kg intraperitoneally for 24 h). RESULTS: In vitro experiments showed that A549 cells were the most sensitive to A. nilotica extract among the tested cell lines. A. nilotica extract inhibited A549 cell proliferation by blocking the cell cycle at the G2/M phase and accumulating apoptotic cells in the sub-G0/G1 phase in A549 cells. In vivo experiments showed that MNU induced positive and negative genotoxicity in bone marrow cells and spermatocytes, respectively. Negative genotoxicity was observed in A. nilotica extract-treated groups only. However, A. nilotica extract (800 mg/kg) remarkably increased comet tail formation in bone marrow cells. Unexpectedly, the absence of antigenotoxicity was observed in three cotreated groups with A. nilotica extract and MNU compared with the MNU-treated group. Astonishingly, cotreatment with MNU and A. nilotica extract at a dose above 200 mg/kg remarkably increased micronucleus and comet tail formation in bone marrow cells compared with the MNU-treated group. CONCLUSIONS: A. nilotica extract possessed anticancer activity with relative genotoxic effects at high doses.

Toxicological assessment of enzyme-treated Zizania latifolia extract: Oral toxicology and genotoxicity in rats.

Zizania latifolia Turcz. has long been used as a food source in Southeast Asia. The grains, stems, and leaves of Z. latifolia and its major component, tricin, have also been studied to determine their biological activities. Previously, we hydrolyzed the aerial part of Z. latifolia using an enzyme mixture to maximize the tricin content of the Z. latifolia extract. However, the safety of enzyme-treated Z. latifolia extract (ETZL; DermaNiA™) has not yet been determined. In this study, we performed an in vivo 90-day repeated-dose evaluation and genotoxicity study to assess the toxicological potential of ETZL. EZTL did not exhibit genotoxicity in the bacterial reverse mutation test, in vitro chromosomal aberration assay, or in vivo micronucleus test. Moreover, no changes in body weight or hematological and serum biological parameters were observed in male or female rats under high-dose EZTL treatment (5000 mg/kg body weight (bw)/day) for 90 days with a 4-week recovery period. Significant changes were noted in the forestomach, kidneys, and adrenal glands in the test groups, but these changes, or tendency for recovery, were not observed in the recovery group. Based on these data, the no adverse effect level was determined to be 1250 mg/kg bw/day in rats.

Antimicrobial, Antigenotoxicity, and Characterization of Calotropis procera and Its Rhizosphere-Inhabiting Actinobacteria: In Vitro and In Vivo Studies.

Calotropis procera (C. procera) is a wild shrub that is a medicinal plant found in abundance throughout Saudi Arabia. In this study, we investigated the phytochemical composition and antigenotoxic properties of the ethanolic extract of C. procera, in addition to the antimicrobial activity of the plant and its rhizospheric actinobacteria effects against pathogenic microorganisms. Soil-extract medium supplemented with glycerol as a carbon source and starch-casein agar medium was used for isolation of actinobacteria from rhizosphere. From the plant, a total of 31 compounds were identified using gas chromatography/mass spectrometry (GC-MS). The main components were α-amyrin (39.36%), lupeol acetate (17.94%), phytol (13.32%), hexadecanoic acid (5.55%), stigmasterol (3.16%), linolenic acid (3.04%), and gombasterol A (2.14%). C. procera plant extract's antimicrobial activity was investigated using an agar well-diffusion assay and minimum inhibitory concentration (MIC) against six pathogenic microbial strains. The plant extract of C. procera was considered significantly active against Staphylococcus aureus, Klebsiella pneumonia, and Escherichia coli, with inhibition zones of 18.66 mm, 21.26 mm, and 21.93 mm, respectively. The plant extract was considered to be a moderate inhibitor against Bacillus subtilis, with MIC ranging from 0.60-1.50 mg/mL. On the other hand, the isolated actinobacteria were considered to be a moderate inhibitor against S. aureus (MIC of 86 µg/mL), and a potent inhibitor, strain CALT_2, against Candida albicans (MIC of 35 µg/mL). The 16S rRNA gene sequence analysis showed that the potential strains belonged to the genus Streptomyces. The effect of C. procera extract against cyclophosphamide (CP)-induced genotoxicity was examined by evaluating chromosome abnormalities in mouse somatic cells and DNA fragmentation assays. The current study revealed that oral pretreatment of C. procera (50, 100, and 200 mg/kg b.w.) for 1, 7, and 14 days to cyclophosphamide-treated animals significantly reduced chromosomal abnormalities as well as DNA fragmentation in a dose-dependent manner. Moreover, C. procera extract had antimicrobial and antigenotoxic effects against CP-induced genotoxicity.

Anticlastogenic, antimutagenic, and cytoprotective properties of Orthosiphon stamineus ethanolic leaves extract.

Orthosiphon stamineus (O.S) is widely consumed for its medidcinal value including anti-inflammatory, anti-infective, and diuretic properties. The present study evaluates the cytoprotective, anti-mutagenic, and anticlastogenic efficacies of standardized extract of Orthosiphon stamineus. Normal liver cell line (WRL68) exposed to hydrogen peroxide and serum-deprived media as insults to evaluate cytoprotective and glutathione activation activities of (Et. O. s). Salmonella typhimurium TA98 and TA100 exposed to different concentrations of (Et. O. s). The influence of Et. O. s on mitotic, replicative indices as well as chromosomal aberration (CA) and sister chromatid exchange (SCE) induced in human peripheral blood lymphocytes by mitomycin C (MMC). The Et. O.s proved to be a potent scavenger for hydrogen peroxide and other free radicals in serum-depraved media, which showed to stimulate glutathione production in liver cells line. Moreover, it did not induce mutations in S. typhimurium subspecies TA98 and TA100. The standardized extract exhibited powerful antimutagenic activities as verified against both 2-nitrofluorene and sodium azide in S. typhimurium TA98 and TA100 cells, respectively. Cytogenetic tests showed high concentrations of Et. O. s to reduce the values of mitotic and replicative indices without any accompanying side effects, such as chromosomal abnormalities or SCE. To ameliorate MMC effects, pretreatment with the extract proofed to be efficient protocol. These data suggests that O. stamineus extract could be useful as cytoprotective, antimutagenic, and anticlastogenic efficacies, which owes to its potent chemoprevention, antioxidant, and glutathione activation properties.

Environmental Risk Characterization of an Antiretroviral (ARV) Lamivudine in Ecosystems.

Antiretroviral drugs for the treatment of human immunodeficiency virus (HIV) and other viral infections are among the emerging contaminants considered for ecological risk assessment. These compounds have been reported to be widely distributed in water bodies and other aquatic environments, while data concerning the risk they may pose to unintended non-target species in a different ecosystem (environment) is scanty. In South Africa and other developing countries, lamivudine is one of the common antiretrovirals applied. Despite this, little is known about its environmental impacts as an emerging contaminant. The present study employed a battery of ecotoxicity bioassays to assess the environmental threat lamivudine poses to aquatic fauna and flora. Daphnia magna (filter feeders), the Ames bacterial mutagenicity test, Lactuca sativa (lettuce) germination test, and the Allium cepa root tip assay were conducted, testing lamivudine at two concentrations (10 and 100 µg/L), with environmental relevance. The Daphnia magna toxicity test revealed a statistically significant response (p << 0.05) with a mortality rate of 85% on exposure to 100 µg/L lamivudine in freshwater, which increased to 100% at 48-h exposure. At lower concentrations of 10 µg/L lamivudine, 90% and 55% survival rates were observed at 24 h and 48 h, respectively. No potential mutagenic effects were observed from the Ames test at both concentrations of lamivudine. Allium cepa bioassays revealed a noticeable adverse impact on the root lengths on exposure to 100 µg/L lamivudine. This impact was further investigated through microscopic examination, revealing some chromosomal aberration in the exposed Allium cepa root tips. The Lactuca sativa bioassay showed a slight adverse impact on both the germination rate of the seeds and their respective hypocotyl lengths compared to the control. Overall, this indicates that lamivudine poses an ecological health risk at different trophic levels, to both flora and fauna, at concentrations previously found in the environment.

Detection and characterization of refractory organic and inorganic pollutants discharged in biomethanated distillery effluent and their phytotoxicity, cytotoxicity, and genotoxicity assessment using Phaseolus aureus L. and Allium cepa L.

The color effluent discharged by alcohol distilleries comprises very high pollution loads due to the plethora of refractory chemicals even after anaerobic treatment and causing adverse effects to the environment. The present study aimed to examine the phytotoxic, cytotoxic, and genotoxic potential of the identified refractory organic and inorganic pollutants discharged in bio-methanated distillery effluent (BMDE). Physico-chemical analyses revealed that BMDE retains high BOD, COD, TDS along with heavy metals like Fe (572.64 mg L-1), Mn (4.269 mg L-1), Cd (1.631 mg L-1), Zn (2.547 mg L-1), Pb (1.262 mg L-1), (Cr 1.257 mg L-1), and Ni (0.781 mg L-1) beyond the permissible limits for effluent discharge. GC-MS analysis revelaed the presence of hexadecanoic acid, TMS ester; octadecanoic acid, TMS ester; 2,3 bis[(TMS)oxy]propyl ester; stigmasterol TMS ether; ß-sitosterol TMS ester; hexacosanoic acid; and tetradecanoic acid, TMS ester as major refractory organic pollutants, which are listed as potential endocrine disruptor chemicals (EDCs) as per USEPA. Furthermore, phytotoxicity assessment with Phaseolus aureus L. showed the toxic nature of BMDE as it inhibited various seedling growth parameters, seed germination, and suppression of α-amylase activity in seed germination experiment. Moreover, genotoxicity and cytotoxicity evaluation of the discharged BMDE evidenced in root-tip meristematic cells of Allium cepa L. where chromosomal aberration such as disturbed metaphase, c-mitosis, laggard chromosomes, sticky chromosomes, prolonged prophase, polyploid cells, and apoptotic bodies etc. were observed. Thus, this study's results suggested that BMDE discharged without adequate treatment poses potential risk to environment and may cause a variety of serious health threats in living beings upon exposure.

Evaluation of oral toxicity and genotoxicity of Achyranthis Radix extract.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Achyranthes bidentata Blume, Achyranthis Radix (AR), is used as a traditional medicine ingredient in East Asia. It has anti-inflammatory, anti-oxidative, and anti-diabetic activities. AIM OF THE STUDY: In the present study, we aimed to evaluate the oral toxicity and genotoxicity of single-dose and 4-week repeated-doses of AR hot water extract (ARE), under the good laboratory practice principles. MATERIALS AND METHODS: For oral toxicity studies, SD rats (n = 5 per sex and group) were administered ARE at concentrations of 500, 1000, and 2000 mg/kg/day once (single dose) or once per day for 4 weeks (repeated dose). The non-clinical genotoxicity study consisted of bacterial reverse mutation using Escherichia coli (WP2 uvrA) and Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), in vitro chromosomal aberration test with Chinese hamster lung cells (CHL/IU), and in vivo mouse bone marrow micronucleus test using bone marrow cells collected from male ICR mice (n = 5) that were orally administered ARE. RESULTS: In the single-dose oral toxicity study, mortality and treatment-related changes in body weight were not observed throughout the study, and the lethal dose was estimated to be > 2000 mg/kg in rats. In the 4-week repeated-dose oral toxicity study, ARE did not induce significant changes in body weight, organ weight, food intake, or hematological and serum biochemical parameters in any group. In the bacterial reverse mutation test, ARE did not induce gene mutations in any tested strain. In the chromosomal aberration test, ARE did not cause chromosomal aberrations. The micronucleus test showed no significant increase in the number of micronucleated polychromatic erythrocytes or the mean ratio of polychromatic to total erythrocytes. CONCLUSIONS: These results showed that ARE does not induce oral toxicity and genotoxicity in the in vivo and in vitro test systems.

Genotoxicity of Water Extract from Bark-Removed Rhus verniciflua Stokes.

Rhus verniciflua Stokes (RVS) has been traditionally used as an herbal remedy to support the digestive functions in traditional Korean medicine. Additionally, the pharmacological effects of RVS, including antioxidative, antimicrobial and anticancer activities, have been well-reported. The genotoxicity of RVS, however, is elusive; thus, we evaluated the genotoxicity of RVS without bark (RVX) for safe application as a resource of functional food or a medical drug. To evaluate the genotoxicity of RVX, we used a bacterial reverse mutation test, chromosomal aberration test and comet assay, according to the "Organization for Economic Co-operation and Development" (OECD) guidelines. Briefly, for the reverse mutation test, samples (5000, 1667, 556, 185, 62 and 0 µg/plate of RVX or the positive control) were treated with a precultured strain (TA98, TA100, TA1535, TA1537 or WP2µvrA) with or without the S9 mix, in which RVX partially induced a reverse mutation in four bacterial strains. From the chromosomal aberration test and comet assay, the RVX samples (556, 185, 62, 20 and 0 µg/mL of RVX or the positive control) were treated in a Chinese hamster ovary cell line (CHO-K1 cells) in the conditions of the S9 mix absent or S9 mix present and in Chang liver cells and C2C12 myoblasts, respectively. No chromosomal aberrations in CHO-K1 or DNA damage in Chang liver cells and C2C12 myoblasts was observed. In conclusion, our results suggest the non-genotoxicity of RVX, which would be helpful as a reference for the safe application of bark-removed Rhus verniciflua Stokes as functional raw materials in the food, cosmetics or pharmaceutical fields.

Residual pollutants in treated pulp paper mill wastewater and their phytotoxicity and cytotoxicity in Allium cepa.

Discharged pulp and paper mill wastewater (PPMW) were collected near M/s K. R. pulp and papers Limited, Shahjahanpur, India. Chemical analysis of the wastewater showed high BOD (3653-4180 mg L-1) and COD (17,890-19100 mg L-1) values from two different sampling sites. The levels of total phenol were in the range of 389-432 mg L-1; nitrogen (125-234 mg L-1), sulfate (1926-2098 mg L-1), chloride (3.12-5.43 mg L-1) and lignin (38,950-39,000 mg L-1) along with various heavy metals (Fe, 87-79; Zn, 34-22; Cu, 3.28-2.57; Cd, 1.90-0.36; Ni, 6-5, and Pb, 41.23-36.54 mg L-1) were above the permissible limits recommended by the CPCB and the USEPA. The BOD/COD ratio was < 0.2 which indicated very low biodegradability of the organic matters present in the effluent. The organometallic complex generated from the pulp and paper industry persists in the environment and might be toxic to aquatic organisms. The organic polymers, lignin, metals and ions present in the PPMW were characterized using SEM, EDAX, FTIR, and UV-VIS spectroscopy. The major pollutants detected in the discharged PPMW included nonacosane, heptacosane, octadecanoic acid, hexadecane, and 6-benzamide- 3- [2- [1-(phenylmethyl)-4-piperidinyl] ethyl]-1, 2-benzisoxazole, as well as a group of plant fatty acids classified as EDCs, and mutagenic pollutants. The cytotoxic and androgenic properties of these complex organics were examined. The seed germination test with Phaseolus mungo and cytotoxicity test with Allium cepa showed that at > 20% concentration of PPMW, α-amylase production was inhibited and chromosomal segregation at metaphase and anaphase during cell division was disturbed, which resulted in c-mitosis, sticky chromosomes, and laggard chromosomes. In addition, SEM of the root of A. cepa showed fissures and fractured tissues of the root cap, probably due to the inhibition of auxins that were responsible for root cap formation. The findings indicated A. cepa as a good test model for examining the DNA damage and cytotoxicity by PPMW, and the discharged effluent should be treated at a tertiary stage for environmental protection.

A study on water quality monitoring of Buddha Nullah, Ludhiana, Punjab (India).

Diverse genotoxic agents, entering the aquatic environment through natural and anthropogenic events, pose serious threats to its biotic components. The present study involves the monitoring of water quality by assessing the genotoxic effects and physico-chemical parameters including heavy metals of 10 surface water samples collected from different locations of Buddha Nullah, a tributary of Sutlej flowing through Ludhiana, Punjab (India). Genotoxicity was evaluated following Allium cepa root chromosomal aberration assay and DNA nicking assay using plasmid (pBR322) whilst the metal (cadmium, chromium, cobalt, copper, lead, nickel and zinc) analysis was conducted using atomic absorption spectrophotometer. All water samples collected from the study area had cobalt and lead content more than the permissible limits (0.04 and 0.01, respectively) recommended by the Bureau of Indian Standards and the World Health Organization. The samples also induced genotoxicity following both bioassays. The water samples collected from Gaunspur (GP), a site approx. 75.53 km upstream of the Sutlej-Buddha Nullah joining point, has shown the maximum genotoxic effect, i.e. 38.62% in terms of per cent total aberrant cells during A. cepa assay and 100% DNA damage during DNA nicking assay. The Pearson correlation indicated that genotoxicity had a significant positive correlation with the content of cobalt (at p ≤ 0.5). During cluster analysis, the samples from 10 sites formed four statistically significant clusters based on the level of pollution that was dependent on two factors like similarity in physico-chemical characteristics and source of pollution at a specific site.

Toxicological safety evaluation of the cultivated Chinese cordyceps.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese cordyceps, a parasitic Thitarodes insect-Ophiocordyceps sinensis fungus complex in the Qinghai-Tibet Plateau, is one of the most valuable traditional Chinese medicines and health food for ameliorating conditions associated with aging and for treating fatigue, night sweats, hyperglycemia, hyperlipidemia, respiratory, renal and liver diseases, and hyposexuality. The natural Chinese cordyceps resource is rare due to its harsh growing environment, limited geographical distribution and global climate warming. Artificial cultivation of Chinese cordyceps has been successfully established to meet its high demand in market. AIMS OF THE STUDY: The present study aims to evaluate the toxicological safety of the cultivated Chinese cordyceps and provide scientific data for subsequent development and utilization of this valuable biological resource. MATERIALS AND METHODS: The Chinese cordyceps was cultivated by mimicking the habitat environment in low-altitude areas and identified by morphological and microscopic characteristics. Its phytochemical profile was determined by the HPLC. Toxicological studies based on the cultivated Chinese cordyceps were conducted, including chromosomal aberration test of Chinese hamster lung (CHL) cells, Ames test, acute toxicity test and micronucleus (MN) test of bone marrow cells. RESULTS: The Chinese cordyceps successfully cultivated in low-altitude areas exhibited the same morphological and microscopic characteristics as natural Chinese cordyceps. The adenosine content was in accordance with the Chinese Pharmacopoeia (2015 Edition). The HPLC fingerprint was determined and five main chromatographic peaks representing uracil, uridine, inosine, guanosine and adenosine were identified. No dose-dependent increase in the rates of chromosomal aberration was detected in the presence or absence of metabolic activation system. Ames test also demonstrated no dose-dependent increase in the number of reversion mutation for five bacterial strains, with or without rat liver microsomal enzyme mixture (S9) metabolic activation, at a quantity range of 128-5000 µg cultivated Chinese cordyceps per plate. The acute toxicity test with mice showed that after 20 g/kg oral administration of cultivated Chinese cordyceps, neither animal death nor any abnormal change in general dissection of various tissues and organs of the animals were found within 14 days. The median lethal dose (LD50) was greater than 5 g/kg, which is regarded as a non-toxic level, and maximum tolerable dose (MTD) of cultivated Chinese cordyceps in ICR mice was more than 20 g/kg. MN test of mouse bone marrow cells indicated no significant differences among each sample dose and the negative control. CONCLUSION: Based on the results from four toxicological tests, it was concluded that the cultivated Chinese cordyceps was classified as non-toxic in one single administration at high doses by intragastric route in mice. This study provides scientific experimental basis for its safety.

A Report of Two Cases in which Kampo Treatment Could Contribute to the Improvement of Quality of Life for Children with Congenital Disorders / 日本東洋医学雑誌

We report two cases in which the quality of life of children with chromosomal aberration improved with Kampo treatment. Case 1 : Six-year-old boy with 5p deletion syndrome. We prescribed kambakutaisoto for the purpose of treating excitability and insomnia. After adding shokenchuto, and further using with yokukansan, the degree of excitement was obviously reduced, and he began to sleep well until morning. Case 2 : Two-year-old boy with chromosome 8 abnormality. He had failed extubation after thoracotomy, thus he needed tracheostomy and gastrostomy. Frequent sputum suction was required every 5 to 15 minutes, and he suffered chronic diarrhea. The number of suctioning was decreased after treatment with bukuryoingohangekobokuto. Moreover, after the addition of shokenchuto, suction became unnecessary for more than 2 hours, and diarrhea also improved. After surgery to close tracheostomy site, he has been receiving ogikenchuto + shoseiryuto + ninjinto and is very well enough to go to a nursery school. Although it is a small improvement not involved in life prognosis, it has contributed to the improvement of the quality of life and the reduction of burden on caregivers. We believe that it is one of the significance of providing Kampo treatment for children.

Investigation of cytotoxicity potential of different extracts of Ziziphus mauritiana (Lam) leaf Allium cepa model.

Previous studies showed that Ziziphus mauritiana is ethnomedicinally beneficial against various diseases, however the cytotoxic and genotoxic effects of this plant have not been well investigated. Therefore this study was undertaken to investigate cytotoxic and genotoxic effects of four different solvents extracts of Ziziphus mauritiana (Lam) leaf at different concentrations (20, 40, 60, 80, and 100 mg/L) using Allium cepa model. The cytotoxicity and genotoxicity parameters evaluated were mitotic index, root tip growth length and chromosomal aberration respectively. The result revealed a decrease in mitotic index percentage (%MI) and a dose dependent decrease in root tip length with increase in extracts concentration for all the extracts studied - with the ethanol extract showing the most significant effect in mitotic index. Furthermore, the effective concentrations (EC50) obtained were 81.30, 52.01, 90.68 and 112.30 mg/l for aqueous, ethanol, ethyl acetate and hexane extract respectively. Finally chromosomal aberrations such as vagrant chromosome, c-mitosis, bridged anaphase, sticky telophase were also observed in all four extracts and the percentage chromosomal aberration were observed to decrease with increased concentrations of extracts. Therefore based on the result obtained in this study it may be concluded that the plant (Ziziphus mauritiana (Lam)) extracts are cytotoxic and genotoxic in nature and the observed decrease in percentage chromosomal aberration may be as a result of antimutagenic bioactive principles present in the plant extracts. Hence care must be taken in its consumption and use in folk medicine.

Evaluation of the biosafety potentials of methanol extracts/fractions of Tapinanthus bangwensis and Moringa oleifera leaves using Allium cepa model.

Medicinal plants are considered two-edged swords. They are pharmacologically beneficial and sometimes toxic when abused. The health benefit of medicinal plant is due to their abundant phytochemical endowment. The present study is aimed at evaluating the biosafety potentials of methanol extracts/fractions of Tapinanthus bangwensis (T. bangwensis) and Moringa oleifera (M. oleifera) leaves using Allium cepa (A. cepa) model. Cytotoxicity assay was carried out by A. epa model. The cytotoxicity parameters studied were number of dividing cells, percentage mitotic index (% MI), root tip growth length and chromosomal aberrations. The phytochemistry was determined by UV-spectrophotometry while 2,2-diphenyl-1-picrylhydrazine (DPPH) and nitric oxide (NO) were used to assay antioxidant activity. The A. cepa assay result showed that the inhibitory effect of M. oleifera on root tip growth length was higher compared to T. bangwensis at 60-100mg/100ml concentrations. Decreased number of dividing cells and percentage mitotic index as concentrations increased observed indicate cytotoxicity however the acetone fractions were most cytotoxic. The frequently occurring chromosomal aberrations observed were vagrant, bridged, attached and sticky chromosomes while C-mitosis and binuclear chromosomes were not observed. Therefore cytotoxic effect was significant at 100mg/100ml. The phytochemical screening of methanol extracts/fractions of T. bangwensis and M. oleifera leaves revealed the presence of alkaloids, flavonoids, phenolics, saponins and tannins however saponins were significantly abundant in both plants compared to others. Alkaloid content was found to be low but was not detected in the acetone fractions of the plants. Methanol extracts/fractions of the plants' leaves exhibited antioxidant activities but was more prominent in the ethylacetate fraction of T. bangwensis and acetone fraction of M. oleifera. In conclusion, administration at 100mg/100ml could be unsafe for the biological system.

DNA damages induced by both endotoxin and exotoxin produced by cyanobacteria.

Cyanobacteria are prokaryotes involved in the contamination of aquatic environments since they release toxins that are highly potent and dangerous for living organisms. Prokaryotes produce endo and exotoxins, among others. Exotoxins are highly toxic, while endotoxins have milder toxic effects. The present study evaluated the cytotoxicogenetic potency of both toxins studying them in different concentrations of cyanobacterial biomasses (1 µg/L, 1.5 µg/L, 2 µg/L), to assess the amount of exotoxin present in the cultured medium in which the cyanobacteria were grown. For this evaluation, we used an extract taken from the medium in a concentration of 10%. Our results showed that genotoxic and mutagenic changes in Allium cepa could be observed in all of the varying concentrations of biomass (endotoxin action) and also in the medium induced with exotoxin. Even at low concentrations, these toxins were highly effective at triggering changes in the DNA molecules of organisms exposed to them. This information is highly significant when considering environmental contamination caused by cyanobacteria blooms, since the results of this study show that these toxins may not only kill organisms when found in high concentrations, but also induce mutations when found in low concentrations. Since these mutations are expressed later on in the organisms, it is impossible to associate the observed effect with the event that induced the damage.

Genotoxicity study of nano Al2O3, TiO2 and ZnO along with UV-B exposure: An Allium cepa root tip assay.

The present effort aims to investigate the cytotoxic and genotoxic impact of three widely used nanoparticles (ZnO, TiO2 and Al2O3) on root cells of Allium cepa as a test organism. The root tip of Allium cepa were treated with three different concentrations (0.1 10 and 100 mg/L) of the above-mentioned NPs and the observations were recorded after proper growth of root under both nanoparticle solutions and UV-B exposure in combined conditions and separately. The parameters such as mitotic index, various forms of chromosomal aberrations, various reactive oxygen species (ROS) generation such as superoxide radical (O-2·), hydrogen peroxide, hydroxyl radical (·OH), lipid peroxidation and bio-uptake of nanoparticles were assessed. The results revealed that for all the three nanoparticles, mitotic index (MI) was highly reduced in comparison to control. Among the three nanoparticles, the MI value of TiO2 was 59.5% at 0.1 mg/L. Chromosomal aberration data suggest that nano Al2O3 exhibited disturbed metaphase at 0.1 mg/L, and abnormal anaphase and sticky metaphase at 10 and 100 mg/L, respectively. Similarly, lagged metaphase and anaphase with multiple chromatin bridges were recorded for both nano ZnO and nano TiO2 at 0.1 mg/L. But, nonsignificant (p > 0.05) results were recorded between only nano metal oxide and UV-B along with nano metal oxide. ROS generation data revealed that ZnO is more active under UV-B than TiO2 and Al2O3. The cellular deformation and the existence of metal in A. cepa under nano ZnO, TiO2 and Al2O3 treatment were evaluated by Scanning Electron Micrograph (SEM) and X-ray fluorescence (XRF) study, respectively. It may safely be concluded that with respect to chromosomal aberration and mitotic index, out of the three nanoparticles, Al2O3 is the most severe at higher concentrations and nano ZnO shows lowest mitotic index under UV-B exposure.

Aqueous extract of Forsythia viridissima fruits: Acute oral toxicity and genotoxicity studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Forsythia viridissima fruit, one of Forsythiae Fructus (FF) is widely used in traditional medicine to treat diverse diseases-related clinical symptoms, including fever, pain, vomiting, nausea, and abscess. However, the safety of FF has not been fully assessed. AIM OF THE STUDY: In this study, we evaluated the acute oral toxicity and genotoxic potential of an aqueous extract of Forsythia viridissima fruits (EFVF). MATERIALS AND METHODS: For an acute oral toxicity test, male and female SD rats (n = 5) orally received a single dose of 5000 mg/kg EFVF. The genotoxic potential of EFVF was evaluated with a battery of tests, including an in vitro bacterial reverse mutation test using five mutant strains of Salmonella typhimurium (TA100, TA1535, TA98, TA1537) and Escherichia coli (WP2 uvrA), an in vitro chromosomal aberration test using Chinese hamster lung (CHL/IU) cells, and an in vivo micronucleus test using bone marrow cells in male ICR mice that were orally administered EFVF. All tests were completed in compliance with Organization for Economic Cooperation and Development guidelines and/or regional regulatory standards for toxicity tests. RESULTS: In the acute oral toxicity test, the animals did not show any significant mortality and body weight changes for 14 days following a single dose of EFVF at 5000 mg/kg. There was no evidence of genotoxicity of EFVF based on the results of the in vitro bacterial reverse mutation test (up to 5000 µg/plate), the in vivo micronucleus test (up to 5000 mg/kg), and the in vitro chromosomal aberration test (1100-2500 µg/mL). CONCLUSIONS: We found that EFVF is safe with regard to acute toxicity in rats as well as genotoxicity such as mutagenesis or clastogenesis under the present experimental conditions. These results might support the safety of EFVF as a potential therapeutic material for the traditional use or pharmaceutical development.

Hydroalcoholic Myrciaria dubia (camu-camu) seed extracts prevent chromosome damage and act as antioxidant and cytotoxic agents.

The camu-camu seeds, which comprehend about 20% of the fruit weight, is discarded without taking benefit of their chemical components and potential application by the industry. In the current study, we characterized the phenolic composition, the in vitro chemical antioxidant effects, cytotoxic activity, and the inhibition of induced-cisplatin chromosomal aberrations of five camu-camu seed extracts obtained with different proportions of water (H2O) and ethyl alcohol (EtOH). The 50% H2O + 50% EtOH was the most promising extract because it presented higher total phenolic content (4802 mg GAE/100 g), antioxidant capacity (DPPH = 3694 mg AAE/100 g; FRAP = 6604 mg AAE/100 g; FCRC = 4918 mg GAE/100 g) and inhibited the cell growth of four cancer cell lines (GI50 = 7.49 µg GAE/mL A549; 13.3 µg GAE/mL Caco-2; 15.57 µg GAE/mL HepG2 and 14.89 µg GAE/mL HCT8) without cytotoxic effects against normal cells (GI50 IMR90 > 43.2 µg GAE/mL). The cytotoxic effects presented high correlation with the (-)-epicatechin and methylvescalagin contents, while gallic and 2,5-dihydroxybenzoic acids were associated with cytoprotective effects of HCT8 cancer cell line. The 50% H2O + 50% EtOH extract also presented protective effect by decreasing 37% of the induced-cisplatin chromosomal breaks index, suggesting its antimutagenic potential, which may be associated to its antioxidant and cytotoxic activities.