Safety assessment and toxicological profiling of a novel combinational sunprotective dermal formulation containing melatonin and pumpkin seed oil

Ultraviolet (UV) radiation exposure has been known to cause irreparable damages to human skin. The daunting risk of UV radiation exposure faced by military personnel led to the development of a sunscreen formulation which has superior sun protection factor combined with the ability to counteract reactive oxygen species. The present work deals with the preclinical safety evaluation of the sunscreen formulation comprising of four US FDA approved UV filters; namely avobenzone, octinoxate, oxybenzone, titanium dioxide along with melatonin and pumpkin seed oil, via OECD protocols of assessing acute oral and dermal toxicity; skin sensitizing; skin irritating; ocular irritating and genotoxic potential. Both oral and dermal LD₅₀ values were found to be ˃2000 mg/kg body weight in adult Wistar albino rats using acute dermal and oral toxicity tests. The sunscreen formulation was found to be non-sensitizing to the skin of guinea pigs and non-irritating to both skin and eyes of rabbits. The sunscreen formulation was also found to be non-mutagenic which was affirmed by a battery of genotoxicity and muagenicity assays. The results obtained from this preclinical study indicated that the sunscreen formulation is non toxic and safe in animal models. This study along with additional preclinical evaluations may serve as a basis for considering the formulation as a potential candidate for further trials to establish its efficacy, tolerability and applicability.     

Black cohosh extracts and powders induce micronuclei,a biomarker of genetic damage,in human cells

Black cohosh extract (BCE) is a widely used dietary supplement marketed to women to alleviate symptoms of gynecological ailments, yet its toxicity has not been well characterized. The National Toxicology Program (NTP) previously reported significant increases in micronucleated erythrocytes in peripheral blood of female Wistar Han rats and B6C3F1/N mice administered 15–1,000 mg BCE/kg/day by gavage for 90 days. These animals also developed a dose‐dependent nonregenerative macrocytic anemia characterized by clinical changes consistent with megaloblastic anemia. Both micronuclei (MN) and megaloblastic anemia can arise from disruption of the folate metabolism pathway. The NTP used in vitro approaches to investigate whether the NTP's test lot of BCE, BCEs from various suppliers, and root powders from BC and other cohosh species, were genotoxic in general, and to gain insight into the mechanism of action of BCE genotoxicity. Samples were tested in human TK6 lymphoblastoid cells using the In Vitro MicroFlow® MN assay. The NTP BCE and a BC extract reference material (XRM) were tested in the MultiFlow^® DNA Damage assay, which assesses biomarkers of DNA damage, cell division, and cytotoxicity. The NTP BCE and several additional BCEs were tested in bacterial mutagenicity assays. All samples induced MN when cells were grown in physiological levels of folic acid. The NTP BCE and BC XRM produced activity patterns consistent with an aneugenic mode of action. The NTP BCE and five additional BCEs were negative in bacterial mutagenicity tests. These findings show that black cohosh preparations induce chromosomal damage and may pose a safety concern. Environ. Mol. Mutagen. 59:416–426, 2018. © 2018 Published 2018. This article is a US Government work and is in the public domain in the USA.     

Diet‐induced obesity increases the frequency of Pig‐a mutant erythrocytes in male C57BL/6J mice

Obesity increases the risk of a number of chronic diseases in humans including several cancers. Biological mechanisms responsible for such increased risks are not well understood at present. Increases in systemic inflammation and oxidative stress, endogenous production of mutagenic metabolites, altered signaling in proliferative pathways, and increased sensitivity to exogenous mutagens and carcinogens are some of the potential contributing factors. We hypothesize that obesity creates an endogenously mutagenic environment in addition to increasing the sensitivity to environmental mutagens. To test this hypothesis, we examined two in vivo genotoxicity endpoints. Pig‐a mutant frequencies and micronucleus frequencies were determined in blood cells in two independent experiments in 30‐week old male mice reared on either a high‐fat diet (60% calories from fat) that exhibit an obese phenotype or a normal‐fat diet (10% calories from fat) that do not exhibit an obese phenotype. Mice were assayed again at 52 weeks of age in one of the experiments. N‐ethyl‐N‐nitrosourea (ENU) was used as a positive mutation control in one experiment. ENU induced a robust Pig‐a mutant and micronucleus response in both phenotypes. Obese, otherwise untreated mice, did not differ from non‐obese mice with respect to Pig‐a mutant frequencies in reticulocytes or micronucleus frequencies. However, such mice, had significantly higher and sustained Pig‐a mutant frequencies (increased 2.5‐3.7‐fold, p < 0.02) in erythrocytes as compared to non‐obese mice (based on measurements collected at 30 weeks or 30 and 52 weeks of age). This suggests that obesity, in the absence of exposure to an exogenous mutagen, is itself mutagenic. Environ. Mol. Mutagen. 57:668–677, 2016. © 2016 Wiley Periodicals, Inc.     

Time in hemodialysis modulates the levels of genetic damage in hemodialysis patients

It is assumed that hemodialysis treatment can diminish the levels of genetic damage in circulating lymphocytes by cleaning the blood of uremic toxins that cause oxidative stress. However, the hemodialysis process by itself may also induce genomic damage by producing reactive oxygen species (ROS). We conducted a follow‐up study in a group of 70 hemodialysis patients followed for a mean time of 15 months. We investigated the effect of exposure time in hemodialysis on the levels of genetic damage in peripheral blood lymphocytes using the micronucleus assay. In addition, genetic damage after in vitro irradiation with 0.5 Gy was also analyzed to evaluate changes in radiosensitivity. Our results showed that, at the end of the study, there was a decrease in both the basal levels of genetic damage (9.9 ± 1.0 vs. 7.6 ± 0.7) and radiosensitivity values (38.5 ± 3.0 vs. 27.6 ± 2.4). We conclude that hemodialysis procedures may act as an ameliorating factor reducing the genetic damage present in chronic kidney disease patients. Environ. Mol. Mutagen. 55:363–368, 2014. © 2014 Wiley Periodicals, Inc.     

Effects of indirubin and isatin on cell viability,mutagenicity, genotoxicity and BAX/ERCC1 gene expression

Context: Indigofera suffruticosa Miller (Fabaceae) and I. truxillensis Kunth produce compounds, such as isatin (ISA) and indirubin (IRN), which possess antitumour properties. Their effects in mammalian cells are still not very well understood.

Objective: We evaluated the activities of ISA and/or IRN on cell viability and apoptosis in vitro, their genotoxic potentials in vitro and in vivo, and the IRN- and ISA-induced expression of ERCC1 or BAX genes.

Materials and methods: HeLa and/or CHO-K1 cell lines were tested (3 or 24?h) in the MTT, Trypan blue exclusion, acridine orange/ethidium bromide, cytokinesis-blocked micronucleus (CBMN) and comet (36, 24 and 72?h) tests after treatment with IRN (0.1 to 200?μM) or ISA (0.5 to 50?μM). Gene expression was measured by RT-qPCR in HeLa cells. Swiss albino mice received IRN (3, 4 or 24?h) by gavage (50, 100 and 150?mg/kg determined from the LD₅₀ – 1?g/kg b.w.) and submitted to comet assay in vivo.

Results: IRN reduced the viability of CHO-K1 (24?h; 5 to 200?μM) and HeLa cells (10 to 200?μM), and was antiproliferative in the CBMN test (CHO-K1: 0.5 to 10?μM; HeLa: 5 and 10?μM). The drug did not induce apoptosis, micronucleus neither altered gene expression. IRN and ISA were genotoxic for HeLa cells (3 and 24?h) at all doses tested. IRN (100 and 150?mg/kg) also induced genotoxicity in vivo (4?h).

Conclusion: IRN and ISA have properties that make them candidates as chemotherapeutics for further pharmacological investigations.     

The application of the cytokinesis-block micronucleus assay on peripheral blood lymphocytes for the assessment of genome damage in long-term residents of areas with high radon concentration

Estimating the effects of small doses of ionising radiation on DNA is one of the most important problems in modern biology. Different cytogenetic methods exist to analyse DNA damage; the cytokinesis-block micronucleus assay (CBMN) for human peripheral blood lymphocytes is a simple, cheap and informative cytogenetic method that can be used to detect genotoxic-related markers. With respect to previous studies on radiation-induced genotoxicity, children are a poorly studied group, as evidenced by the few publications in this area. In this study, we assessed radon genotoxic effects by counting micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in the lymphocytes of children who are long-term residents from areas with high radon concentrations. In the exposed group, radon was found to cause significant cytogenetic alterations. We propose that this method can be employed for biomonitoring to screen for a variety of measures.     

Application of the buccal micronucleus cytome assay and analysis of PON1Gln192Arg and CYP2A6*9(−48T>G) polymorphisms in tobacco farmers

Tobacco is a major Brazilian cash crop. Tobacco farmers apply large amounts of pesticides to control insect growth. Workers come into contact with green tobacco leaves during the tobacco harvest and absorb nicotine through the skin. In the present study, micronucleus frequency, cell death, and the frequency of basal cells were measured in tobacco farmers using the buccal micronucleus cytome assay (BMCyt), in parallel with measurement of blood butyrylcholinesterase (BChE) and nicotine levels. Polymorphisms in PONIGln192Arg and CYP2A6*9(?48T>G) were evaluated to verify the relationship between genetic susceptibility and the measured biomarkers. Peripheral blood and buccal cell samples were collected from 106 agricultural workers, at two different crop times (during pesticide application and leaf harvest), as well as 53 unexposed controls. BMCyt showed statistically significant increases in micronuclei, nuclear buds, and binucleated cells among exposed subjects in differentiated cells, and in micronuclei in basal cells. In addition, the exposed group showed higher values for condensed chromatin, karyorrhectic, pyknotic, and karyolitic cells, indicative of cell death, and an increase in the frequency of basal cells compared to the unexposed control group. A slight difference in mutagenicity using the BMCyt assay was found between the two different sampling times (pesticide application and leaf harvest), with higher micronucleus frequencies during pesticide application. Elevated cotinine levels were observed during the leaf harvest compared to the unexposed controls, while BChE level was similar among the farmers and controls. PONIGln192Arg and CYP2A6*9(?48T>G) polymorphisms were associated with DNA damage induced by pesticides and cell death. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

The role of urinary pH in o‐phenylphenol‐induced cytotoxicity and chromosomal damage in the bladders of F344 rats

o‐Phenylphenol (OPP) is a widely used fungicide and antibacterial agent that at high doses has been shown to cause bladder cancer in male F344 rats. The mechanisms underlying OPP‐induced bladder carcinogenicity remain unclear but it has been proposed that a non‐enzymatic pH‐dependent autoxidation of phenylhydroquinone (PHQ), a primary metabolite of OPP, may be a key step in OPP‐induced rat bladder carcinogenesis. To investigate this mechanism and to provide insights into the potential human health relevance of OPP‐induced cancer, a series of in vitro and in vivo experiments were conducted. In human lymphoblastoid TK‐6 cells and rat bladder epithelial NBT‐II cells, strong increases in cytotoxicity were seen at a constant concentration of PHQ by increasing the buffer pH as well as by increasing concentrations of PHQ at a constant pH. In in vivo studies, male rats were administered OPP (4,000 and 8,000 ppm) in a diet supplemented with either 1% ammonium chloride or 3% sodium bicarbonate to produce acidic and alkaline urinary pH, respectively. Significant increases in cell proliferation as detected by 5‐bromo‐2′‐deoxyuridine incorporation and micronucleus formation were seen in the bladder cells of OPP‐treated rats with neutral or alkaline urinary pH but not in animals with the acidified urine. The results from these in vitro and in vivo studies provide support for the autoxidation hypothesis of bioactivation, and provide additional evidence that urinary pH can significantly influence the genotoxicity and carcinogenicity of this important agent. Environ. Mol. Mutagen. 57:210–219, 2016. © 2016 Wiley Periodicals, Inc.     

脾胃湿热证胃炎患者舌苔脱落细胞中微核细胞及P53表达

[目的]探讨脾胃湿热证和脾气虚证慢性浅表性胃炎患者舌苔脱落细胞微核细胞和P53表达的意义.[方法]用Feulgen法和免疫细胞化学法显示正常对照者及脾胃湿热和脾气虚患者舌苔脱落细胞中微核细胞、P53表达.[结果]脾胃湿热组和脾气虚组微核细胞的反应强度及出现频率均高于正常对照组(均P＜0.05);脾气虚组的P53阳性反应高于正常对照组,差异有统计学意义(P＜0.05).[结论]微核细胞在两患者组的舌苔脱落细胞内反应强度及出现频率较高,提示有较多的分裂畸变细胞;而P53的表达与微核细胞的出现有关,两者可能代表了证候发展不同的过程.     

Earthworm biomarker responses on exposure to commercial cypermethrin

Cypermethrin is a synthetic pyrethroid insecticide used worldwide in agriculture, home pest control, disease vector control, and food safety. It accumulates in soil. Therefore, traces of cypermethrin may frequently appear in vegetables grown in contaminated soil. There is a push now to develop biomarkers as early warning indicators of environmental pollution. In this study, DNA damage (tail DNA%, tail length, and olive tail moment), the micronucleus, neutral red retention (NRR) time, and pinocytic adherence ability of coelomocytes were investigated in Pheretima peguana earthworms exposed to cypermethrin in filter paper tests. The NRR time of earthworm coelomocytes decreased significantly at a concentration of 3.5 × 10^?3 µg · cm^?2 (1/100 LC₅₀) after 48 h exposure, with a highly negative correlation with cypermethrin concentration. Pinocytic adherence ability of coelomocytes also declined significantly at a cypermethrin concentration of 3.5 × 10^?2 µg · cm^?2 (1/10 LC₅₀). The DNA damage to earthworm coelomocytes (tail DNA%, tail length, and olive tail moment) increased considerably at the highest concentration (3.5 × 10^?1 µg · cm^?2) although the correlation between tail DNA% and cypermethrin concentration was low. Thus, physiological biomarkers were more sensitive than the genotoxic effects in earthworms exposed to commercial cypermethrin. Although a suite of earthworm biomarkers could be used to evaluate cypermethrin terrestrial pollution, the NRR test is easier to conduct and a more sensitive indicator. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 597–606, 2015.