Safety evaluation of 5-hydroxytryptophan and S-(2-aminoethyl)isothiouronium bromide hydrobromide on rodent lungs.

OBJECTIVES: During the past few decades, various compounds have been researched for their potential as radioprotectants, and many of them were found to be safe and effective in several preclinical models. However, many of these compounds were found to have serious adverse effects when evaluated in clinical settings, thereby making them unsuitable for human applications. 5-hydroxytryptophan (5-HTP) and S-(2-aminoethyl) isothiouronium bromide hydrobromide (AET) act in a synergistic fashion to promote radioprotection. The present study primarily emphasizes the safety of fixed dose of 5-HTP + AET in the lungs of C57BL/6 mice, a well-known model used in drug safety studies. MATERIALS AND METHODS: Post-administration of the combination of HTP+AET at specific time points, blood and bronchoalveolar lavage fluid (BALF) were collected for the analysis of inflammatory and oxidative stress markers of the lungs. Thereafter, the mice were sacrificed and the lungs were dissected out, weighed, and fixed in formalin for histopathological studies. RESULTS: The inflammatory biomarkers: tumor necrosis factor-alpha and interleukin-10 and oxidative stress biomarkers: 8-isoprostane and 8-hydroxy-2'-deoxyguanosine were found to have normal levels in blood and BALF in both control and treatment groups, which was further supported by normal histological findings. In addition, other endpoints such as food and water intake were found to be within normal limits. CONCLUSION: The present safety study reflects that the combination has no adverse effects on the lungs of the experimental mouse. Further, evaluation in higher mammals including nonhuman primates is essential prior to validation of the safety of the combination in humans.

Differentiated and exponentially growing HL60 cells exhibit different sensitivity to some genotoxic agents in the comet assay.

The aim of this study was to investigate the effect of the cell differentiation status on the sensitivity to genotoxic insults. For this, we utilized the comet assay to test the DNA damage after treatment with 5 different substances with different mechanism of action in human promyelocytic HL60 cells with or without cell differentiation. A 4-hour MMS treatment induced a significant and concentration-dependent increase in DNA damage for both differentiated and undifferentiated cells, but the difference in sensitivity was only significant at the highest concentration. A 4-hour doxorubicin treatment did not induce DNA damage in differentiated HL60 cells, while it did in undifferentiated cells with its highest tested concentration. A one-hour etoposide treatment caused significant increase in DNA damage concentration dependently in both cell variants. This DNA damage was significantly higher in undifferentiated HL60 cells with several tested concentrations of etoposide. The treatment with the oxidizing substances hydrogen peroxide and potassium bromate yielded significant DNA damage induction in both undifferentiated and differentiated cells with no difference according to the differentiation status. Doxorubicin and etoposide are known to inhibit topoisomerase II. The activity of this enzyme has been shown to be higher in undifferentiated actively proliferating cells than in differentiated cells. This may be of relevance when exposures to topoisomerase-inhibiting compounds or the genotoxicity of compounds with unknown mechanism of action are assessed in routine testing.

Potential impurities in drug substances: Compound-specific toxicology limits for 20 synthetic reagents and by-products, and a class-specific toxicology limit for alkyl bromides.

This paper provides compound-specific toxicology limits for 20 widely used synthetic reagents and common by-products that are potential impurities in drug substances. In addition, a 15 µg/day class-specific limit was developed for monofunctional alkyl bromides, aligning this with the class-specific limit previously defined for monofunctional alkyl chlorides. Both the compound- and class-specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals. Inhalation-specific toxicology limits were also derived for acrolein, formaldehyde, and methyl bromide because of their localized toxicity via that route. Mode of action was an important consideration for a compound-specific toxicology limit. Acceptable intake (AI) calculations for certain mutagenic carcinogens assumed a linear dose-response for tumor induction, and permissible daily exposure (PDE) determination assumed a non-linear dose-response. Several compounds evaluated have been previously incorrectly assumed to be mutagenic, or to be mutagenic carcinogens, but the evidence reported here for such compounds indicates a lack of mutagenicity, and a non-mutagenic mode of action for tumor induction. For non-mutagens with insufficient data to develop a toxicology limit, the ICH Q3A qualification thresholds are recommended. The compound- and class-specific toxicology limits described here may be adjusted for an individual drug substance based on treatment duration, dosing schedule, severity of the disease and therapeutic indication.

Toxic effects of ionic liquid 1-octyl-3-methylimidazolium bromide on the antioxidant defense system of freshwater planarian, Dugesia japonica.

The activities of antioxidant enzymes and the levels of glutathione (GSH) and malondialdehyde (MDA) were determined when freshwater planarian Dugesia japonica was exposed to different concentrations of 1-octyl-3-methylimidazolium bromide ([C8mim]Br) for one, three, and five days. The results showed that superoxide dismutase (SOD) activity began to increase in all treated groups after three days of exposure, while catalase (CAT) activity was inhibited after the first day, but increased notably on the fifth day except for the lowest concentration group. The activity of glutathione peroxidase (GPX) was induced from the first day of exposure and increased significantly after five days in all treated groups. During the experiment, the levels of intracellular GSH in all treated groups were higher than that of the control group. Changes in MDA suggest that [C8mim]Br is toxic to D japonica and may result in lipid peroxidation in planarian. Our results also indicate that GPX as well as GSH seem to be more sensitive biomarkers of oxidative stress compared with SOD and CAT.

Cytotoxicity of 1-dodecyl-3-methylimidazolium bromide on HepG2 cells.

We evaluated the cytotoxicity of 1-dodecyl-3-methylimidazo-lium bromide ([C12mim][Br]) on HepG2 cells and its influence on plasma membrane permeability. The results showed that [C12mim][Br] inhibited HepG2 cell growth and decreased cell viability in a concentration-depen-dent manner. The results also revealed that [C12mim][Br] exposure induced apoptosis in [C12mim][Br]-treated HepG2 cells. In addition, the results showed that [C12mim][Br] increased membrane permeability in HepG2 cells. These results suggest that plasma membrane permeability may be responsible for apoptosis induced by [C12mim][Br] in HepG2 cells.

Toxicity of ionic liquids prepared from biomaterials.

In search of environmentally-friendly ionic liquids (ILs), 14 were prepared based on the imidazolium, pyridinium and choline cations, with bromide and several amino acids as anions. Good yields were obtained in the synthesis of pyridinium ILs and those prepared from choline and amino acids. Four of the ILs synthesized from choline and the amino acids arginine, glutamine, glutamic acid and cystine are described here for the first time. The toxicity of the synthesized ILs was checked against organisms of various levels of organization: the crustacean Artemia salina; Human cell HeLa (cervical carcinoma); and bacteria with different types of cell wall, Bacillus subtilis and Escherichia coli. The toxicity was observed to depend on both the cation and anion. Choline-amino acid ILs showed a remarkable low toxicity to A. salina and HeLa cell culture, ten times less than imidazolium and pyridinium ILs. None of ionic liquids exhibited marked toxicity to bacteria, and the effect was 2-3 orders of magnitude smaller than that of the antibiotic chloramphenicol.

In vitro cytotoxicities of ionic liquids: effect of cation rings, functional groups, and anions.

In vitro cytotoxicities were measured for ionic liquids (ILs) containing various cations and anions using the MCF7 human breast cancer cell line. We measured the cytotoxicities of ionic liquids containing the cations pyridinium, pyrrolidinium, piperidinium, or imidazolium with various alkyl chain lengths, and the anions bromide, bis(trifluoromethanesulfone)imide (Tf(2)N), trifluoromethylsulfonate (TfO), or nonafluoromethylsulfonate (NfO). Three new hydrophobic, task-specific ionic liquids (TSILs), namely, [MBCNPip](+)[Tf(2)N](-), [MPS(2)Pip](+)[Tf(2)N](-), and [MPS(2)Pyrro](+)[Tf(2)N](-) designed for metal-ion extraction were also evaluated. IC(50) values of the ionic liquids toward the MCF7 cells ranged from 8 microM to 44 mM. The toxicity depended significantly on the nature of the cations and anions, especially when the cations contained a long side chain. TSILs studied in this work were less toxic than the classical ILs.

Total synthesis of phenanthroindolizidine alkaloids (+/-)-antofine, (+/-)-deoxypergularinine, and their dehydro congeners and evaluation of their cytotoxic activity.

Due to their limited natural abundance and significant biochemical effects, we synthesized the alkaloids (+/-)-antofine (1a), (+/-)-deoxypergularinine (1b), and their dehydro congeners (2 and 3) starting from the corresponding phenanthrene-9-carboxaldehydes. We also evaluated their in vitro cytotoxic activity. Compounds 1a and 1b showed significant potency against various human tumor cell lines, including a drug-resistant variant, with EC(50) values ranging from 0.16 to 16ng/mL. Structure-activity correlations of these alkaloids and some of their synthetic intermediates were also ascertained. The non-planar structure between the two major moieties, phenanthrene and indolizidine, plays a crucial role in the cytotoxic activity of phenanthroindolizidines. Increasing the planarity and rigidity of the indolizidine moiety significantly reduced potency. A methoxy group at the 2-position (1a) was more favorable for cytotoxic activity than a hydrogen atom (1b).

Mutagenicity of nitrobenzyl derivatives: potential bioreductive anticancer agents.

Ortho-, meta- and para-nitrobenzyl bromides, alcohols, ethers and esters were synthesised and tested for their mutagenicity toward Salmonella typhimurium TA100, TA100NR (nitroreductase deficient) and TA98 in absence of S9 mix and in TA100 with S9 mix. Compounds of the ortho- and meta-series were non mutagenic with and without S9 mix. Except for the alcohol and ether, the compounds of the para-series were mutagenic in TA100 with activity sequence propionate > butyrate > benzoate > acetate > bromide and this specific activity was reduced considerably by S9 mix. The Ames Salmonella test system does not seem to be an appropriate model to evaluate mutagenicity of o-nitrobenzyls. However, further work is in progress to test all the compounds for mutagenicity in mammalian system.

A possible role for cell proliferation in potassium bromate (KBrO3) carcinogenesis.

Accumulation of alpha 2u-globulin and induction of cell proliferation were examined in kidneys of rats exposed to KBrO3, KBr or NaBrO3 in their drinking water. Hyaline droplets observed after KBrO3 or NaBrO3 administration to male rats were specifically immunostained for alpha 2u-globulin. Increases in cell proliferation were found in the proximal tubules of male rats given KBrO3 or NaBrO3 but not KBr for 2, 4, and 8 weeks. No such change was evident in KBrO3-treated female rats or the distal tubules of any treated animal. The concordance between hyaline droplet accumulation and increased cell turnover suggests that KBrO3- and NaBrO3-induced cell replication in kidneys of male rats may result from alpha 2u-globulin nephropathy. Considering the fact that KBrO3 has genotoxic potential involving oxidative stress, we hypothesize that the induced cell proliferation might predominantly play an additive role in its carcinogenesis. Furthermore, the present data, showing similar effects of NaBrO3 on the rat kidney, are of direct significance to its risk assessment.

Elevated serum bromide concentrations following repeated halothane anaesthesia in a child.

A 20-month-old child received 25 brief halothane general anaesthetics over a five-week period to allow cranial irradiation treatments for a posterior fossa ependymoma. Personality change during the last week of the treatment protocol raised the question of possible bromide intoxication. Serum bromide concentrations, using a gold chloride assay technique, were monitored at that time, and at four- and six-week intervals thereafter. Serum bromide concentrations demonstrated a four-fold change during this period ranging from peak levels of 2.2 mEq.L-1 (176 micrograms.kg-1) during the fifth week of treatment decreasing to less than 0.5 mEq.L-1 (less than 40 micrograms.ml-1) six weeks following the end of treatments. This demonstrates the possibility for repetitive, short halothane exposures to result in elevations of serum bromide and the potential of bromide intoxication in paediatric neuro-oncology patients.

Toxicity and carcinogenicity of potassium bromate--a new renal carcinogen.

Potassium bromate (KBrO3) is an oxidizing agent that has been used as a food additive, mainly in the bread-making process. Although adverse effects are not evident in animals fed bread-based diets made from flour treated with KBrO3, the agent is carcinogenic in rats and nephrotoxic in both man and experimental animals when given orally. It has been demonstrated that KBrO3 induces renal cell tumors, mesotheliomas of the peritoneum, and follicular cell tumors of the thyroid. In addition, experiments aimed at elucidating the mode of carcinogenic action have revealed that KBrO3 is a complete carcinogen, possessing both initiating and promoting activities for rat renal tumorigenesis. However, the potential seems to be weak in mice and hamsters. In contrast to its weak mutagenic activity in microbial assays, KBrO3 showed relatively strong potential inducing chromosome aberrations both in vitro and in vivo. Glutathione and cysteine degrade KBrO3 in vitro; in turn, the KBrO3 has inhibitory effects on inducing lipid peroxidation in the rat kidney. Active oxygen radicals generated from KBrO3 were implicated in its toxic and carcinogenic effects, especially because KBrO3 produced 8-hydroxydeoxyguanosine in the rat kidney. A wide range of data from applications of various analytical methods are now available for risk assessment purposes.

Two-year oral chronic toxicity and carcinogenicity study in rats of diets fumigated with methyl bromide.

A chronic toxicity and carcinogenicity study was conducted by feeding diets containing 80, 200 or 500 ppm total bromine following fumigation with methyl bromide, a diet containing 500 ppm potassium bromide, or a basal diet alone to groups of 60 male and 60 female Fischer (F344) rats for up to 2 yr. Ten males and ten females from each group were killed after wk 52 and 104 for urinalysis, haematology, blood biochemistry and pathology. Rats that were killed in extremis or found dead during the study and all rats that survived to the end of the study were also subjected to pathological examinations. In rats fed the diets fumigated with methyl bromide there were no marked toxic changes, except for a slight depression of body-weight gain from wk 60 onwards in males of the 500-ppm group, and tumour incidence was unaffected. Rats given a diet containing potassium bromide did not show any treatment-related changes. It was concluded that residues of up to 500 ppm total bromine in diets fumigated with methyl bromide are not carcinogenic in F344 rats of either sex and that the maximum no-effect level is 200 ppm (6.77 mg total bromine/kg body weight/day) in males. The maximum no-effect level could not be determined in females.

Injurious effect of the eosinophil peroxide-hydrogen peroxide-halide system and major basic protein on human nasal epithelium in vitro.

Tissue injury is observed in allergic and nonallergic eosinophilic rhinitis, but the mechanism of this injury is unclear. Because eosinophils are prominent in biopsy specimens in these conditions, we hypothesized that they may participate in the injury process. Initially, we developed techniques to isolate and purify human nasal epithelial cells from turbinate biopsies to use as target cells for eosinophil granule products. Primary cultures from explants were characterized by electron microscopy and indirect immunofluorescence with a panel of primary monoclonal and polyclonal antibodies. These studies revealed the homogeneity of the cells and confirmed their epithelial nature. Cultured nasal epithelial cells were then exposed to either purified human eosinophil peroxidase, bromide, and glucose plus glucose oxidase, as a continuous source of hydrogen peroxide, or eosinophil major basic protein. Neither eosinophil peroxidase alone nor glucose plus glucose oxidase in the absence of eosinophil peroxidase were injurious, but the combined addition of eosinophil peroxidase, glucose/glucose oxidase, and bromide produced marked target cell lysis. This effect was time- and eosinophil peroxidase dose-dependent. Catalase and azide significantly inhibited the lysis of these cells, suggesting the eosinophil peroxidase-catalyzed products of halide oxidation mediated this form of injury. The addition of purified human eosinophil major basic protein also caused dose- and time-dependent lysis of the nasal epithelial cells but required longer incubation periods to effect injury. We hypothesize that the eosinophil peroxidase-hydrogen peroxide-halide system and major basic protein may injure the nasal epithelium in inflammatory conditions such as allergic and nonallergic eosinophilic rhinitis.

Acute toxicity studies of aluminium compounds: antidotal efficacy of several chelating agents.

Four aluminum compounds--nitrate, chloride, sulphate and bromide--were administered orally and intraperitoneally to rats and mice. The LD50-values (14 days) were determined. The majority of deaths occurring during the first four days. The clinical and physical signs appearing after intoxication include among other lethargy, decreased locomotor activity, piloerection, weight loss and perorbital bleeding. After 14 days no alterations in liver and renal functions were detected in the animals which received intraperitoneally the LD50-values of aluminum nitrate as a single dose. Aluminum concentrations were highest in liver and spleen. No histopathological lesions could be observed. To compare the efficacies of nine chelating agents on the toxicity of aluminum in mice, the therapeutic index and the therapeutic effectiveness of each chelating agent have been calculated. Malic, succinic, oxalic and malonic acids showed the best results with malic and succinic acids being the most effective. Deferoxamine mesylate (DFOA), sodium salicylate, L-cysteine and citric acid were not so effective as antidotes for acute aluminum toxicity. Aurin tricarboxylic acid (ATCA) should not be used due to its high toxicity.

Efectos toxicológicos producidos por la ingesta crónica de aceites vegetales bromados / Toxicological effects induced by the chronic intake of brominated vegetable oils

Se estudiaron las alteraciones de parámetros lipídicos a nivel tisular y plasmático en un grupo de ratas macho de la cepa Wistar, alimentadas con una dieta normal de laboratorio suplementada con aceites vegetales-oliva, girasol-bromados (0.1 g/100 g de dieta), durante 15 semanas, comparando los resultados obtenidos con un grupo control. El primer grupo presentó niveles estadísticamente elevados de triglicéridos en corazón y soleus, y de colesterol total y esterificado en músculo cardíaco, acompañados por un descenso en los niveles plasmáticos de colesterol total y de la fracción de lipoproteínas de alta densidad (HDL-colesterol). Algunas de estas anormalidades fueron compartidas por ratas alimentadas con la dieta normal de laboratorio suplementada con 0.5 g de aceites vegetales bromados/100 g dieta. Los niveles hepáticos de triglicéridos, proteínas totales y glucógeno, así como la curva de crecimiento y la ingesta calórica de los animales que fueron alimentados con una dosis de aceites bromados de 0.1 g/100 g de dieta, fueron similares a los del grupo control. En síntesis, los efectos toxicológicos observados durante la ingesta crónica de dietas suplementadas con dosis relativamente bajas de aceites bromados, señalan la necesidad de emprender un estudio bioquímico más exhaustivo de los mismos. Ello es imperativo para establecer los niveles máximos con que estos agentes podrían ser utilizados sin riesgo de producir alteraciones biológicas considerables

Effect of sodium bromide on endocrine parameters in the rat as studied by immunocytochemistry and radioimmunoassay.

Male rats were fed a normal or sodium bromide-enriched diet for 4 or 12 weeks. Sodium bromide concentrations were 0, 20, 75, 300, 1200 and 19,200 mg/kg diet. At the end of the experiments the pituitary gland, thyroid and testes were examined by histopathological and immunocytochemical techniques, while serum hormone levels were established by radioimmunoassay. Histopathological examination revealed an activation of the thyroid and a decreased spermatogenesis in the testes in the highest dose group. Using immunocytochemical techniques a decrease was noted in the amount of thyroxine in the thyroid. No effect was found in growth hormone-producing cells in the pituitary gland, while immunoreactivity for thyroid-stimulating hormone and for adrenocorticotropic hormone was increased. The concentration of thyroxine, testosterone and corticosterone in the serum appeared to be decreased. Due to feedback regulation, the pituitary gland was stimulated to produce and release thyroid-stimulating hormone, follicle-stimulating hormone, adrenocorticotropic hormone and insulin, whereas the release of growth hormone was suppressed. Most of these changes were restricted to rats on the highest treatment level. It is concluded that sodium bromide, at least in high doses, directly disturbs the function of the thyroid, testes and adrenals.