Weak Genotoxic Activity Research Articles

Semicarbazide Hydrochloride as Impurity in Drug Substances: a Validated LC-DAD-UV Method for Its Determination in Carbazochrome and Carbazochrome Sodium Sulfonate

Carbazochrome (2-(1,2,3,6-tetrahydro-3-hydroxy-1-methyl-6-oxo-5H-indol-5-ylidene)-hydrazinecarboxamide, CBZ) and carbazochrome sodium sulfonate (5-[(aminocarbonyl)hydrazono]-2,3,5,6-tetrahydro-1-methyl-6-oxo-1H-indole-2-sulfonic acid sodium salt, CSS) are derivatives of adrenochrome currently employed mainly as hemostatic drugs. Since a semicarbazone group is present in both the structures of CBZ and CSS, semicarbazide hydrochloride (SEM) is used, as reagent, in their synthesis. Belonging to the hydrazine family of chemicals, SEM could possess some toxic potential. Accordingly, literature data showed that SEM causes osteolathyrism and induces toxic effects in the cardiovascular and skeletal systems. Moreover, SEM has a weak genotoxic activity in vitro while in vivo available data are not sufficient to state its genotoxicity. Based on these data, the determination of SEM as a synthesis-related impurity in CBZ and CSS drug substances is mandatory. Since there are no publications dealing with the determination of SEM in CBZ and CSS as well as in any other SEM-containing drugs, a new and unprecedented LC-DAD-UV method for its determination as impurity in CBZ and CSS drug substances was developed and validated. Due to the lack of chromophore, semicarbazide was converted into the corresponding UV-absorbing naphthalene-2-carbaldehyde semicarbazone (NCS) before the chromatographic analysis. The complete separation of NCS from the large concentration of the derivatization reagent and the parent drugs CBZ and CSS was accomplished through a liquid–liquid extraction (LLE) step followed by a chromatographic run of 10 min. The method was then successfully validated and its applicability demonstrated by the determination of SEM in real samples of CBZ and CSS.

Photolytic fate and genotoxicity of benzophenone-derived compounds and their photodegradation mixtures in the aqueous environment.

This study investigates the environmental fate of eight benzophenone derivatives (the pharmaceutical ketoprofen, its phototransformation products 3-ethylbenzophenone and 3-acetylbenzophenone, and five benzophenone-type UV filters) by evaluating their photolytic behaviour. In addition, the genotoxicity of these compounds and the produced photodegradation mixtures was studied. Laboratory-scale irradiation experiments using a medium pressure UV lamp revealed that photodegradation of benzophenones follows pseudo-first-order kinetics. Ketoprofen was the most photolabile (t1/2 = 0.8 min), while UV filters were more resistant to UV light with t1/2 between 17 and 99 h. The compounds were also exposed to irradiation by natural sunlight and showed similar photostability as predicted under laboratory conditions. Solar photodegradation experiments were performed in distilled water, lake and seawater, and revealed that photosensitizers present in natural waters significantly affect the photolytic behaviour of the investigated compounds. In this case, the presence of lake water resulted in accelerated photodecomposition, while seawater showed different effects on photodegradation, depending on a compound. Further, it was shown that the transformation products of ketoprofen 3-ethylbenzophenone and 3-acetylbenzophenone were formed under environmental conditions when ketoprofen was exposed to natural sunlight. Genotoxicity testing of parent benzophenone compounds using the SOS/umuC assay revealed that UV filters exhibited weak genotoxic activity in the presence of a metabolic activation system, however the concentrations tested were much higher than found in the environment (≥125 μg mL(-1)). After irradiation of benzophenones, the produced photodegradation mixtures showed that, with the exception of benzophenone that exhibited weak genotoxic activity, all the other compounds tested did not elicit any activity when exposed to UV light.

Genotoxic effects of Ag2S and CdS nanoparticles in blue mussel (Mytilus edulis) haemocytes

The use of functionalised metal sulphide nanoparticles (NPs) for nanoremediation and biomedical application is rapidly increasing, which could lead to significant inputs into the marine environment. The potential impact of some NPs on marine organisms is still poorly understood. In the present paper the genotoxic potential of Ag2S and CdS NPs on Mytilus edulis haemocytes was assessed. MPEG-SH (thiol-terminated methyl polyethylene glycol), was used as capping agent to avoid NPs agglomeration. TEM analysis showed that the Ag2S NPs size was 13±7 nm, whereas CdS quantum dots had an average diameter of 4±1 nm. DNA integrity was evaluated by Comet assay following exposure to increasing concentration series (0.01–10 mg/L). Both silver and cadmium NPs showed genotoxic effects at the highest dose. MPEG-SH was also found to exert a weak genotoxic activity, suggesting that at least part of the genotoxic potential of functionalised NPs on mussel cells might be attributable to the capping agent. These results confirm the genotoxic potential of Ag2S NPs for mussel cells and demonstrated, for the first time, that CdS NPs is genotoxic in a marine organism.

Derivation of a Melamine Oral Reference Dose (RfD) and Drinking-Water Total Allowable Concentration

Due to its high nitrogen content, melamine has been used to adulterate food to increase apparent protein content. In 2008, thousands of Chinese infants consumed reconstituted formula derived from melamine-adulterated milk. Urinary-tract stones (comprised of melamine and uric acid) accumulated in some victims and lead to acute renal failure or death. Premature infants and children (<2 yr) have an increased susceptibility to ingested melamine. Due to incomplete reporting, the human data were inadequate to identify a no-observed-adverse-effect level (NOAEL) for melamine-induced pediatric urolithiasis. Urolithiasis, urinary bladder cystitis, and ulcerations were observed in F344 rats after subchronic or chronic ingestion of melamine at ≥72 mg/kg-d. Bladder epithelial damage was followed by epithelial hyperplasia that progressed to bladder papillomas and carcinomas in male but not female F344 rats or male or female B6C3F1 mice. Short-term assays suggest, at best, weak genotoxic activity, and kinetic data show that melamine is not metabolized. Since reliable exposure information was lacking from the clinical reports, an oral reference dose (RfD) based on urolithiasis in male rats after 13 wk of continuous melamine ingestion was calculated as a 10% benchmark dose (38 mg/kg-d). Incorporation of 10-fold interspecies and intraspecies (for the increased susceptibility of infants) uncertainty factors and a threefold database uncertainty factor (for the lack of immunological, neurological and reproduction toxicity data) yields an oral RfD of 0.13 mg/kg-d. Assuming the 70-kg adult consumes 2 L of drinking water daily, a total allowable concentration of 0.9 mg/L (900 μg/L) was calculated for melamine in drinking water.

Biological assays and noncovalent interactions of pyridine-2-carbaldehyde thiosemicarbazonecopper(II) drugs with [poly(dA–dT)]2, [poly(dG–dC)]2, and calf thymus DNA

The interaction of the Cu(II) drugs CuL(NO(3)) and CuL'(NO(3)) (HL is pyridine-2-carbaldehyde thiosemicarbazone and HL' is pyridine-2-carbaldehyde 4N-methylthiosemicarbazone, in water named [CuL](+) and [CuL'](+)) with [poly(dA-dT)](2), [poly(dG-dC)](2), and calf thymus (CT) DNA has been probed in aqueous solution at pH 6.0, I = 0.1 M, and T = 25 degrees C by absorbance, fluorescence, circular dichroism, and viscosity measurements. The results reveal that these drugs act as groove binders with [poly(dA-dT)](2), with a site size n = 6-7, whereas they act as external binders with [poly(dG-dC)](2) and/or CT-DNA, thus establishing overall electrostatic interaction with n = 1. The binding constants with [CuL'](+) were slightly larger than with [CuL](+). The title compounds display some cleavage activity in the presence of thiols, bringing about the rupture of the DNA strands by the reactive oxygen species formed by reoxidation of Cu(I) to Cu(II); this feature was not observed in the absence of thiols. Mutagenic assays performed both in the presence and in the absence of S9 mix, probed by the Ames test on TA 98, TA 100, and TA 102, were negative. Weak genotoxic activity was detected for [CuL](+) and [CuL'](+), with a significative dose-response effect for [CuL'](+), which was shown to be more cytotoxic in the Ames test and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assays. Methylation of the terminal NH(2) group enhances the antiproliferative activity of the pyridine-2-carbaldehyde thiosemicarbazones.

Application of the margin of exposure (MoE) approach to substances in food that are genotoxic and carcinogenic: Example: Methyleugenol, CASRN: 93-15-2

The weight of evidence points to weak genotoxic activity of methyleugenol, the putative genotoxic carcinogen being 1′-sulphate ester metabolite. Dose response modelling of the data for methyleugenol gave a BMDL10 for male rat liver adenoma or carcinoma (combined) of 7.9 mg/kg-bw/d following adjustment to daily average doses. The MoEs ranged from 100 to 800 depending on the assumptions used in the exposure estimation.

In vitro short-term test evaluation of catecholestrogens genotoxicity

Estrogens are indicated as being the most important etiological factors for the development and progression of breast cancer. The implication of estrogen in breast cancer has been associated mostly with the estrogen receptors that mediate cell proliferation. Evidence also exists to support the hypothesis of a direct role of estrogens as tumor initiators. However, the role of estrogen genotoxicity in breast cancer is still questionable. In this study the genotoxic activity of catecholestrogens and 16α-hydroxy estrone has been investigated by performing Salmonella strain TA98 and TA100 Ames tests, sister chromatide exchange assays (SCE) and micronucleus assays on human peripheral lymphocytes (CBMN and ARA/CBMN). We found a lack of positive results with micronucleus assays, except for 2-hydroxy estradiol (2-OHE 2), which shows a peculiar “bell shaped” trend of micronucleus number versus concentrations. SCE assay suggests weak genotoxic activity of all tested catechol metabolites, except 4-hydroxy estrone (4-OHE 1), which also showed negative results by ARA/CBMN. In this open debate, our results support the hypothesis of a weak genotoxicity, not correlated with the carcinogenetic potential of estrogens.

Genotoxic activity of nitrilotriacetic acid in Chinese hamster cells

Nitrilotriacetic acid (NTA), a chelating agent, was tested for its ability to induce chromosomal damage in Chinese hamster cells. The chemical was shown to exert a weak genotoxic activity increasing the frequency of micronuclei after prolonged treatments. The analysis of kinetochore containing-micronuclei showed that NTA prevailingly induces chromosomal aberrations as compared to chromosome loss in hamster cells. Furthermore, immunostaining with an α-tubulin antibody showed clear alterations in the interphase microtubule network of cells treated for 24 h with 3 mM NTA. The microtubule effects of the chemical may be partly responsible for its cytotoxic effects.

Genotoxicity testing of different types of beverages in the drosophila wing somatic mutation and recombination test

Five wines and one brandy of Spanish origin as well as three herbal teas and ordinary black tea were tested for genotoxicity in the wing Somatic Mutation And Recombination Test (SMART) which makes use of the two recessive wing cell markers multiple wing hairs ( mwh) and flare ( flr 3) on the left arm of chromosome 3 of Drosophila melanogaster. 3-day-old larvae trans-heterozygous for these two markers were fed the beverages at different concentrations and for different feeding periods using Drosophila instant medium. Somatic mutations or mitotic recombinations induced in the cells of the wing imaginal discs give rise to mutant single or twin spots on the wing blade of the emerging adult flies showing either the mwh phenotype or/and the flr phenotype. One of the red wines showed a clear genotoxic activity that was not due to its ethanol content. Two herbal teas ( Urtica dioica, Achillea millefolium) and black tea ( Camellia sinensis) proved to be weakly genotoxic as well. Furthermore, it was shown that quercetin and rutin, two flavonols present in beverages of plant origin, also exhibited weak genotoxic activity in the somatic cells of Drosophila. These results demonstrate that Drosophila in vivo somatic assays can detect the genotoxicity of complex mixtures such as beverages. In particular, it is possible to administer these test materials in the same form as that in which they are normally consumed.

Mutagenic activity of arylnitrenium ions from arylazides — Induction of sister chromatid exchange in mammalian (V79 Chinese hamster) cells

Photolysis of arylazides produces short-lived reactive species, very likely arylnitrenium ions which bind to nucleotides and DNA and produce mutations in Salmonella. The present report shows that arylazides can be photo-activated in mammalian (V79 Chinese hamster) cells and that sister chromatid exchange can thus be induced. Arylazides studied are (in order of decreasing SCE-inducing potency) azido-isoIQ, azido-MeIQ, azido-IQ, azido-MeIQx, azido-PhIP, 6-azido-chrysene, 2-azidofluorene, 4-azidofluorene, 2-azido-naphthalene, 4-azidobi-phenyl, 2-azidobiphenyl, 2,4,6-trimethylphenylazide, phenylazide (inactive). The structure-activity relationships emerging from the data are the same as those found previously in Salmonella. In line with this, a clearcut positive linear correlation was seen between the logarithm of the SCE-inducing potency in V79 cells and the logarithm of the mutagenic potency in Salmonella ( r = 0.955). Therefore, the ultimate reactive species derived from IQ and related heterocyclic aromatic amines are extremely potent genotoxins, not only in a bacterial but also in a mammalian cell. Previous findings of only weak genotoxic activity of IQ and related food mutagens in certain cultured mammalian cells must therefore be reinterpreted as the result of an insufficient activation of these amines in the cells used, possibly because of insufficient acetylation competence.

Investigations on the use of EDTA-permeabilized E. coli cells in liquid suspension and animal-mediated genotoxicity assays

The potential use of EDTA-permeabilized E. coli cells for the investigation of genotoxic effects of compounds with a large molecular configuration in vitro and in animal-mediated differential DNA-repair assays was studied. The indicator for the induction of (repairable) DNA damage was a pair of E. coli K-12 strains (343/765 and 343/753) differing vastly in DNA-repair capacity ( uvr +/ rec + vs. uvrB/ recA). Investigations on the influence of EDTA treatment on the viability of these strains show that during short-term exposure (3 min), the EDTA level should not exceed 0.5 mmole/1 in the pretreatment mix, since at higher concentrations a marginal titer reduction of the repair-deficient strain occurs, thus indicating a weak genotoxic activity of this chelating agent. Comparisons of the results gained in vitro with permeabilized and untreated cells demonstrate that EDTA exposure leads to a substantial enhancement of the sensitivity of the indicator bacteria towards DNA damage induced by B(a)P and N-Ac-2AAF which is essential for the detection of genotoxic activities of these polycyclic aromatic compounds. Experiments to elucidate the possibility of employing EDTA-treated cells in vivo show that following intravenous and oral administration the recovery rates of permeabilized indicator strains from various mouse organs are substantially lower than those found under identical conditions (exposure time 150 min) with untreated strains. Nevertheless enough viable cells can be recovered from liver, spleen, kidneys, lungs and stomach to allow the investigation of organ-specific genotoxicity. It is furthermore noteworthy that exposure of permeabilized indicator cells in control animals (for 150 min) resulted in a marginal reduction of the relative survival of the repair-deficient strain in all organs investigated, whereas with non-treated strains such effects are only detectable after extended exposure periods. The observation of a slightly elevated genotoxic background under in vivo conditions does not prevent the assessment of the organ distribution of genotoxic effects induced by mutagens and/or carcinogens: in the case of B(a)P, intraperitoneal administration to mice in the dose range of 10–50 mg/kg body weight resulted in a pronounced dose-dependent inactivation of the uvrB/ recA cells in the liver. Also in the lungs differential killing effects occurred at the highest dose tested, whereas no genotoxic activities were detectable in stomach, kidneys and spleen of the host animals.

Genotoxic activity of m-nitrobenzaldehyde

m-Nitrobenzaldehyde (MNB) was evaluated for mutagenic activity using the Ames microbial mutagenicity test and for its ability to induce DNA single-strand breaks in rat hepatocytes as measured by alkaline elution. MNB was tested in S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100, both with and without pretreatment with liver microsomes (S9) isolated from rats pretrented with Aroclor 1254. MNB produced 2-fold or greater increases in revertants in TA1538, both with and without S9, and in TA100 with S9 only. A 2-fold increase in revertants was seen in TA98, but only at the highest dose tested which did not produce inhibition of background growth. MNB caused a greater than 3-fold increase in elution slope, with DNA alkaline elution assay, but only at highly cytotoxic doses and, therefore, is not considered genotoxic in this system. It is concluded that MNB possesses weak genotoxic activity.