Liver Comet Assay Research Articles

Improved effect of pumpkin seed oil against the bisphenol-A adverse effects in male mice.

The present study was conducted to evaluate the ameliorative role of pumpkin seed oil (PSO) against potential adverse effects of bisphenol-A (BPA) in male mice. BPA was administered to the mice orally at a dose of 50 mg/kg body weight once a day for 28 successive days. While, PSO was administered to the mice orally at 1 mL/kg b w either before, with or after treatment of BPA, once a day for 28 successive days. The studied parameters were DNA damage evaluation using comet assay in liver and testes cells and micronucleus test in bone marrow; and histopathological examination of liver and testes tissues. Results revealed that BPA induced DNA damage in tested cells and marked histopathological alterations in liver and testes. In contrast, PSO treatments alleviated DNA damage and improved the histopathological alterations in liver and testes tissues. Furthermore, administration of mice with the PSO before BPA treatment was the best regimen in the alleviation of the adverse effects of BPA, followed by administration of PSO after then with treatment of BPA. It can be concluded that PSO may has a protective role against BPA genotoxicity and histopathological alterations in male mice.

Toxicity of naphthalene in the Neotropical Fish Astyanax Lacustris (Characiformes: Characidae) and Geophagus Brasiliensis (Perciformes: Cichlidae)

Polycyclic aromatic hydrocarbons are one of the most important organic pollutants in environmental studies. The aim of this study was to assess the naphthalene acute toxicity in two fish species, Astyanax lacustris (LLcust, 1875) and Geophagus brasiliensis (Quoy & Gaimard, 1824). The fish were exposed to naphthalene (0.005, 0.03, 0.3, and 3 mgL-1) in water and after that the piscine micronucleus test in erythrocytes, comet assay in blood, liver and gill cells, glutathione S–transferase (GST) activity in the liver, and accumulation of naphthalene in the bile were performed. The susceptibility of the two species was similar and naphthalene was not genotoxic in all tested tissues. The liver GST activity may have been responsible for less damage observed in the liver while the highest DNA damage occurred in blood cells. However, low concentrations of naphthalene in water can stimulate apparent benefits, such as less DNA damage, which would be a compensatory response to an imbalance of homeostasis. The naphthalene is absorbed and can accumulate in the gall bladder, a greater accumulation of PAH was observed in A. lacustris, while G. brasiliensis did not differ from the control. The naphthalene concentrations are not genotoxic to the tested species, although they can potentially accumulate into the body.Keywords: Comet assay. Ecotoxicology. Fish. Genotoxicity. Hormesis.

Slight hypercalcemia is not associated with positive responses in the Comet Assay in male rat liver

Maintenance of physiological levels of intracellular and extracellular calcium is essential for life. Increased intracellular calcium levels are involved in cell death (apoptosis and necrosis) and are associated with positive responses in the Comet assay in vitro. In addition, high calcium and vitamin D intakes were reported to induce apoptosis in adipose tissue in obese mice and to increase DNA-migration in the Comet assay. To investigate increased serum concentration of calcium as a potential confounding factor in the regulatory Comet assay in vivo, we induced mild hypercalcemia in male Wistar rats by 3-day continuous intravenous infusion of calcium gluconate and performed the Comet assay in the liver in line with regulatory guidelines.The results of the study showed that mild increases in serum calcium concentration (up to 1.4 times above the concurrent control) and increased urinary calcium concentration (up to 27.8 times above the concurrent control) results in clinical signs like mild tremor, faster respiration rate and decreased activity in a few animals. However, under the conditions of the study, no increase in the %Tail DNA in the Comet assay and no indication of liver damage as determined by histopathological means were observed. Thus, mild increases in plasma calcium did not lead to positive results in a genotoxicity assessment by the Comet assay in the rat liver. This result is important as it confirms the reliability of this assay for regulatory evaluation of safety.

Toxicological assessment of nano and micron-sized tungsten oxide after 28 days repeated oral administration to Wistar rats

Tungsten oxide (WO3) nanoparticles (NPs) are being used in various applications. However, the health consequences of WO3 NPs exposure have not been explored extensively. Hence, the goal of this study was to evaluate the toxicity of WO3 NPs and their microparticles (MPs) after 28days repeated oral administration in Wistar rats. The particles were characterised by transmission electron microscopy (TEM), dynamic light scattering (DLS), laser Doppler velocimetry (LDV), Brunner-Emmett-Teller (BET), X- ray diffraction (XRD), and inductively coupled plasma optical emission spectrometer (ICP-OES). Genotoxicity was determined using comet assay in blood and liver and micronucleus test in bone marrow. Biochemical parameters such as aspartate aminotransferase and alanine aminotransferase in serum and reduced glutathione content, catalase and lipid peroxidation in liver tissue were determined. Histopathological changes in tissues were documented. Biodistribution of tungsten (W) in rat’s blood, urine, feces and tissues were analysed. The mean size of WO3 NPs and MPs by TEM was 52±2.97nm, and 5.73±7.58μm and morphology were spherical in both the particles. DLS of NPs was 195.6nm. XRD and BET data of WO3 NPs and MPs showed a hexagonal and tetragonal crystal structure and surface area of 19.33 and 15.15(m2/g), respectively. The results revealed a significant increase in DNA damage and micronuclei, a difference in biochemical levels and histopathological alterations after exposure to 1000mg/kg dose of WO3 NPs. W biodistribution was detected in all the tissues in a dose and organ-dependent manner in both the particles. The highest amount of W was found in the liver and lowest in the brain of the treated rats. The tested NPs were found to have little toxicity hazard.

Scientific Opinion on Flavouring Group Evaluation 226 Revision 1 (FGE.226Rev1): consideration of genotoxicity data on one α,β-unsaturated aldehyde from chemical subgroup 1.1.1(b) of FGE.19.

The EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids was requested to evaluate the genotoxic potential of one flavouring substance from subgroup 1.1.1(b) of FGE.19 in the Flavouring Group Evaluation 226 (FGE.226). The flavour industry provided genotoxicity studies for the substance 4,5‐epoxydec‐2(trans)‐enal [FL‐no: 16.071]. Based on these data, the Panel concluded in FGE.226 that 4,5‐epoxydec‐2(trans)‐enal did not induce gene mutations in bacterial cells but was positive in an in vitro micronucleus assay, so, 4,5‐epoxydec‐2(trans)‐enal is considered an in vitro genotoxic agent. The negative results obtained in an in vivo micronucleus assay cannot overrule the positive results of the in vitro micronucleus assay with and without S9‐mix due to the lack of demonstration of bone marrow exposure. Following this, the flavour industry has provided plasma analysis of a satellite group of rats treated with 4,5‐epoxydec‐2(trans)‐enal in order to investigate the systemic exposure of animals in the in vivo micronucleus assay. However, the plasma analysis did not provide enough evidence of target tissue exposure. An in vivo Comet assay in rodents was recommended in FGE.226, in order to investigate possible genotoxic effects at the first site of contact (e.g. stomach/duodenum cells) and in the liver. An in vivo Comet assay in liver and duodenum was provided that suggests that 4,5‐epoxydec‐2(trans)‐enal [FL‐no: 16.071] did not induce DNA damage in the duodenum of rats. However, the genotoxic effect observed in vitro was confirmed in the in vivo Comet assay in the liver of rats. The Panel concluded that 4,5‐epoxydec‐2(trans)‐enal [FL‐no: 16.071] does raise a safety concern with respect to genotoxicity and, therefore, it cannot be evaluated according to the Procedure.

Honey and quercetin reduce ochratoxin A-induced DNA damage in the liver and the kidney through the modulation of intestinal microflora

ABSTRACTThis study was conducted to evaluate the interactions between the gut microbiota, ochratoxin A and functional food such as honey and quercetin, and the consequences of these interactions on ochratoxin-induced DNA damage in blood, liver and kidney cells. Honey (2 g kg−1) or Quercetin (50 mg kg−1) was applied to mice by intragastric application every day for 15 days, immediately before ochratoxin treatment (100 µg kg−1). We investigated colonic probiotic bacteria count, β-glucuronidase activity, the alkaline comet assay in blood, liver and kidney, the number of cells in the peritoneal cavity, macrophage spreading index and hematological and biochemical parameters. Honey and QU may reduce ochratoxin-induced DNA damage in the liver and kidney, β-glucuronidase activity and inflammation, partly through increasing the colon Bifidobacteria and Lactobacilli counts. The obtained results suggest that honey and QU counteracted the OTA-induced toxicity due to their bifidogenic activity and antigenotoxic activity.

Оценка генотоксичности парацетамола и его комбинации с верапамилом <i>in vivo</i>

Парацетамол в дозах 30 и 60 мг/кг (внутрь) отдельно или в сочетании с известным комутагеном верапамилом (0,25 – 10 мг/кг, внутрь) при однократном или 5-кратном ежедневном введении не проявил цитогенетической активности в клетках костного мозга мышей. Цитогенетический эффект отсутствовал также после однократного введения парацетамола в дозе 300 мг/кг. В тесте ДНК-комет парацетамол при однократном введении в дозах 30 и 60 мг/кг на срок 3 или 24 ч не проявил ДНК-повреждающей активности в клетках костного мозга, печени, почек, селезенки и головного мозга мышей. В дозе 300 мг/кг при экспозиции в течение 3 ч парацетамол индуцировал ДНК-кометы типа «ghost cells» в клетках печени, что было расценено как проявление гепатотоксического эффекта препарата.

Report on the rat Pig-a assay using an anti-rat erythroid marker HIS49 antibody in a single dose study of 1,2-dimethylhydrazine

As part of a collaborative study in the Mammalian Mutagenicity Study (MMS) Group of the Japanese Environmental Mutagen Society (JEMS), we investigated the in vivo genotoxicity profile of 1,2-dimethylhydrazine (DMH) using a Pig-a assay in total red blood cells (RBC Pig-a assay) or a reticulocyte Pig-a assay (PIGRET assay). We also assessed the genotoxic potential of DMH using both a bone marrow micronucleus test and a liver comet assay as follow-up studies. Single administration of 25, 50, 100mg/kg DMH to male rats did not show time- or dose-related increases in Pig-a mutant frequency (MF) in either the RBC Pig-a or PIGRET assays up to 4 weeks after treatment. The bone marrow micronucleus test under the same dose levels was judged positive, while the liver comet assay was judged inconclusive due to the high number of hedgehogs. Re-evaluation of the rat liver comet assay at lower dose levels (4, 10, and 25mg/kg DMH) showed a dose-related increase in%DNA in tail. Taken together, DMH showed a positive response in both the bone marrow micronucleus test and liver comet assay, while the increases in Pig-a MF in both the RBC Pig-a and PIGRET assays could hardly be detected after single dosing. These results suggest that DMH provides different genotoxicity outcomes depending on the endpoint following acute in vivo dosing.

In vitro and in vivo genotoxicity assessment of the dopamine receptor antagonist molindone hydrochloride.

Molindone hydrochloride is a dihydroindolone neuroleptic with dopamine D2 and D5 receptor antagonist activity. As an integral component of its preclinical safety evaluation, molindone hydrochloride was evaluated in a series of in vitro and in vivo genetic toxicology assays. In the bacterial reverse gene mutation assays employing four Salmonella tester strains (TA98, TA100, TA1535, and TA1537) and the E. coli tester strain WP2uvrA, molindone hydrochloride was negative in all strains, except TA100, in which it induced a positive response (up to 3-fold) in the presence of rat liver S9. With human S9, a small (2-fold), but nonreproducible, increase in revertants was observed in TA100 at the highest concentration of molindone tested (5,000 µg/plate). The mutagenicity was completely abrogated by the addition of glutathione and UDP-glucuronic acid to rat liver S9, suggesting detoxification of the mutagenic metabolite(s) by Phase II conjugation reactions, pathways commonly operational in humans. Molindone hydrochloride did not induce chromosomal aberrations in human lymphocyte cultures, did not elicit a positive response in a rat bone marrow micronucleus test for clastogencity/aneugenicity, and did not give a positive response in the rat liver comet assay for DNA damage. Collectively, the weight of evidence from these studies, combined with a large margin of safety and efficient detoxification through Phase II conjugation supports the interpretation that molindone hydrochloride does not pose a genotoxic risk to humans at the anticipated clinical dose levels.

Genotoxicity of a thiosulfonate compound derived from Allium sp. intended to be used in active food packaging: In vivo comet assay and micronucleus test

Components of Allium species have antimicrobial and antioxidant properties. A commercial Allium sp. extract (Proallium AP®), of which the main constituent is propyl thiosulphinate oxide (PTSO), is being used in the development of active food packaging. In previous in vitro genotoxicity studies, PTSO, in the presence of metabolic activation, increased the appearance of micronuclei (MN). We assessed the genotoxicity PTSO in rats following oral administration (doses: 5.5, 17.4, and 55mg/kg). The comet assay in liver and stomach (OECD 489) and the MN assay in bone marrow (OECD 474) were carried out. After necropsy, histopathological examinations of the liver and the stomach were performed. The results revealed no in vivo genotoxicity and the histopathological analysis showed only slight modifications, such as increased glycogen storage in the liver and a degenerative process in stomach, with vacuolization of cell membranes, only at the highest dose. Therefore, the present work confirms that this compound is not genotoxic and could be considered as a natural alternative to synthetic preservatives used in the food packaging industry.

Genotoxicity and mutagenicity induced by acute crack cocaine exposure in mice

Context: Crack cocaine is an illicit drug derived from cocaine, in which use and abuse have increased around the world, especially in developing countries. Objectives: The aim of this study was to evaluate genomic damage in multiple organs of mice following acute exposure to crack cocaine. For this purpose, single cell gel (comet) assay in peripheral blood, liver, kidney, and brain cells was performed and micronucleus test for bone narrow and liver cells was also made in this setting. Material and methods: A total of 20 C57BL/10 male mice were distributed into four groups, as follows: 0, 4.5, 9, and 18 mg/kg b.w. of crack cocaine dissolved to 1% dimethyl sulfoxide by intraperitoneal (i.p.) route. All animals were sacrificed 24 h after i.p. injection. Results: The results showed that crack cocaine induced DNA damage in peripheral blood, and brain cells for higher doses used as depicted by single cell gel (comet) assay data. Analysis of kidney cells showed no genetic damage for all groups tested. The number of micronucleated cells did not increase after crack cocaine exposure in bone narrow or liver cells. Conclusion: In summary, crack cocaine is a genotoxic agent in peripheral blood, liver, and brain cells but not mutagenic in multiple organs of mice.

Scientific Opinion on Flavouring Group Evaluation 208 Revision 1 (FGE.208Rev1): Consideration of genotoxicity data on representatives for 10 alicyclic aldehydes with the α,β‐unsaturation in ring / side‐chain and precursors from chemical subgroup 2.2 of FGE.19

The EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids was requested to evaluate the genotoxic potential of flavouring substances from subgroup 2.2 of FGE.19 in the Flavouring Group Evaluation 208 Revision 1 (FGE.208Rev1). The Flavour Industry has provided additional genotoxicity studies on p-mentha-1,8-dien-7-al [FL-no: 05.117], the representative substance for FGE.19 subgroup 2.2. This substance was tested in vivo in a combined micronucleus assay in bone marrow and Comet assay in liver and duodenum. It did not induce any increase in micronucleated polychromatic erythrocytes of the bone marrow of male rats in the micronucleus test and it did not induce DNA damage in duodenum of the same animals as analysed by the Comet assay. The Comet assay performed in liver shows a positive result and therefore the Panel concluded that p-mentha-1,8-dien-7-al [FL-no: 05.117] is genotoxic in vivo and that, accordingly, there is a safety concern for its use as flavouring substance. Since p-mentha-1,8-dien-7-al [FL-no: 05.117] is representative for the nine remaining substances of this subgroup 2.2 (p-mentha-1,8-dien-7-ol [FL-no: 02.060], myrtenol [FL-no: 02.091], myrtenal [FL-no: 05.106], 2,6,6-trimethyl-1-cyclohexen-1-carboxaldehyde [FL-no: 05.121], myrtenyl formate [FL-no: 09.272], p-mentha-1,8-dien-7-yl acetate [FL-no: 09.278], myrtenyl acetate [FL-no: 09.302], myrtenyl-2-methylbutyrate [FL-no: 09.899] and myrtenyl-3-methylbutyrate [FL-no: 09.900]), there is a potential safety concern for these substances.

Alkaline comet assay in liver and stomach, and micronucleus assay in bone marrow, from rats treated with 2-acetylaminofluorene, azidothymidine, cisplatin, or isobutyraldehyde

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined the ability of the assay to determine the genotoxicity of 2-acetylaminofluorene (AAF), azidothymidine (AZT), cisplatin (CPN), and isobutyraldehyde (IBA) in liver and glandular stomach of male Sprague–Dawley rats. Rats were given oral doses of test compound or control once daily for three days. High dose levels were approximately maximum tolerated doses and were based on preliminary range-finding studies. Tissues were harvested 3h after the final dose (48h after the initial dose). A bone marrow micronucleus assay (MN) was also conducted on the rats treated with AZT, CPN, and IBA. Acute toxic effects of treatment were determined primarily through histomorphologic analysis of liver and stomach but also by body weight and serum liver enzyme changes. The comet assay was conducted on fresh tissue preparations but frozen samples from two studies were also assayed. Statistically significant dose-related differences in comet % DNA in tail were found in liver and stomach for the genotoxin AZT and in liver for the genotoxin CPN, but not in liver or stomach for the non-genotoxin IBA. Statistically significant differences in % DNA in tail were measured in liver for the low and mid dose of the genotoxin AAF, but not the high dose. The comet assays of frozen liver suspensions from CPN- and AAF-treated rats yielded comparable results to the assays of fresh preparations. There were no indications of significant toxicity induced by any treatment. The micronucleus assay was positive for CPN and AZT and negative for IBA. In conclusion, the in vivo comet assay is capable of detecting genotoxic effects of a variety of chemicals and may fill an important role in the genotoxicity test battery.

Experiences with the in vivo and in vitro comet assay in regulatory testing.

The in vivo comet assay has recently been implemented into regulatory genotoxicity testing of pharmaceuticals with inclusion into the ICH S2R1 guidance. Regulatory genotoxicity testing aims to detect DNA alterations in form of gene mutations, larger scale chromosomal damage and recombination and aneuploidy. The ICH S2R1 guideline offers two options of standard batteries of tests for the detection of these endpoints. Both options start with an AMES assay and option 1 includes an in vitro mammalian cell assay and an in vivo micronucleus assay in rodent, whereas option 2 includes an in vivo micronucleus assay in bone marrow in rodent and a second in vivo assay in a second tissue with a second endpoint. The test recommended as second in vivo test is the comet assay in rat liver. The in vivo comet assay is considered as mature enough to ensure reliable detection of relevant in vivo genotoxicants in combination with the micronucleus test in bone marrow and the AMES assay. Although lots of research papers have been published using the in vitro comet assay, the in vitro version has not been implemented into official regulatory testing guidelines. A survey of the years 1999-2014 revealed 27 in vivo comet assays submitted to BfArM with market authorisation procedures, European and national advice procedures and clinical trial applications. In three procedures, in vitro comet assays had been submitted within the genetic toxicology packages.

In vivo genotoxicity of Ginkgo biloba extract in gpt delta mice and constitutive androstane receptor knockout mice.

The National Toxicology Program study of Ginkgo biloba extract (GBE), a herbal supplement, reported concerns regarding genotoxicity and clear evidence of hepatocarcinogenicity and liver hypertrophy in mice. To clarify the genotoxicity of GBE in vivo, we performed reporter gene mutation assay using gpt delta mice. We also used a combined liver comet assay and bone marrow micronucleus assay using C3H-derived constitutive androstane receptor knockout (CARKO) and wild-type mice. No remarkable increases in gpt or Spi(-) mutation frequencies were observed in DNA extracted from the livers of gpt delta mice that had been exposed to GBE up to 2000 mg/kg bw/day. In the comet and micronucleus assays, no statistically significant increases in positive cells were observed at doses up to 2000 mg/kg bw/day of GBE in either mouse genotype. The present study provides clear evidence that GBE is not genotoxic in vivo. Our results indicate that GBE-induced hepatocarcinogenesis in mice occurs through a non-genotoxic mode of action.

Influence of experimental conditions on data variability in the liver comet assay

The in vivo comet assay has increasingly been used for regulatory genotoxicity testing in recent years. While it has been demonstrated that the experimental execution of the assay, for example, electrophoresis or scoring, can have a strong impact on the results; little is known on how initial steps, that is, from tissue sampling during necropsy up to slide preparation, can influence the comet assay results. Therefore, we investigated which of the multitude of steps in processing the liver for the comet assay are most critical. All together eight parameters were assessed by using liver samples of untreated animals. In addition, two of those parameters (temperature and storage time of liver before embedding into agarose) were further investigated in animals given a single oral dose of ethyl methanesulfonate at dose levels of 50, 100, and 200 mg/kg, 3 hr prior to necropsy. The results showed that sample cooling emerged as the predominant influence factor, whereas variations in other elements of the procedure (e.g., size of the liver piece sampled, time needed to process the liver tissue post-mortem, agarose temperature, or time of lysis) seem to be of little relevance. Storing of liver samples of up to 6 hr under cooled conditions did not cause an increase in tail intensity. In contrast, storing the tissue at room temperature, resulted in a considerable time-dependent increase in comet parameters.

Cytotoxicity and genotoxicity assessment of silver nanoparticles in mouse

Silver nanoparticles (AgNPs) are among the most commercially used nanomaterials and their toxicity and genotoxicity are controversial. Although many in vitro studies have been conducted to evaluate the genotoxicity of AgNPs, in vivo genotoxicity studies on the nanomaterials are limited. Given the unique physicochemical properties and complex pharmacokinetics behavior of nanoparticles (NPs), in vivo genotoxicity assessment of AgNPs is badly needed. In this study, the clastogenicity and mutagenicity of AgNPs with different sizes and coatings were evaluated using mouse micronucleus (MN) assay, Pig-a assay and Comet assay. Five 7-week-old male B6C3F1 mice per group were treated with 5 nm polyvinylpyrrolidone (PVP)-coated AgNPs at a single dose of 0.5, 1.0, 2.5, 5.0, 10.0 or 20.0 mg/kg body weight (bw) via intravenous injection for both the MN and Pig-a assays; or with 15–100 nm PVP- or 10–80 nm silicon-coated AgNPs at a single or 3-day repeated dose of 25.0 mg/kg bw for the MN assay and Comet assay in mouse liver. Inductively coupled plasma mass spectrometry (ICP-MS) and transmission electron microscopy (TEM) analyses indicated that AgNPs reached the testing tissues (bone marrow for the MN and Pig-a assays and liver for the Comet assay). Although there was a reduction of reticulocytes in the PVP-coated AgNPs-treated animals, indicating cytotoxicity of the AgNPs, none of the treatments resulted in a significant increase of either mutant frequencies in the Pig-a gene or the percent of micronucleated reticulocyte over the concurrent controls. However, both the PVP- and silicon-coated AgNPs induced oxidative DNA damage in mouse liver. These results demonstrate that the AgNPs can reach mouse bone marrow and liver, and generate cytotoxicity to the reticulocytes and oxidative DNA damage to the liver.

Assessment of the Genotoxic Potential of Azidothymidine in the Comet, Micronucleus, and Pig-a Assay

The genotoxic potential of azidothymidine (Zidovudine, AZT), chosen as a model compound for nucleotide analogs, was comprehensively assessed in vivo for gene mutation, clastogenicity, and DNA breakage endpoints. Male Wistar rats were treated by oral gavage over 7 days with AZT at dose levels of 2×0 (control), 2×250, 2×500, and 2×1000mg/kg/day with a final single dose given on day 8. DNA damage was then evaluated with the comet assay in liver, stomach, and peripheral blood and with the micronucleus test in bone marrow and peripheral blood (by flow cytometry) in the same animals. After a treatment-free period of upto 42 days, the Pig-a gene mutation assay was performed in peripheral blood of the high-dose animals. In the comet assay as well as the micronucleus test, AZT caused a considerable dose-dependent increase in DNA damage in all tissues evaluated and was highly cytotoxic to bone marrow and peripheral blood cells. These data are well in line with published results. Surprisingly, AZT did not significantly increase the number of Pig-a mutant cells. We speculate that two factors likely contributed to this negative result: a predominance of large deletions caused by AZT, and the relatively low statistical power of the first-generation scoring method used for this study.

Evaluation of the Mutagenicity and Genotoxicity of Arrabidaea chica Verlot (Bignoneaceae), an Amazon Plant with Medicinal Properties

Arrabidaea chica Verlot (Bignoniaceae) is an important folk medicine plant native to the Amazon region and used to treat anemia, hemorrhage, inflammation, intestinal colic, hepatitis, and skin affections. Although studies showed its therapeutic properties, little knowledge regarding genotoxic properties of this plant is available. The aim of this study was to determine the potential mutagenic and genotoxic/antigenotoxic effects of an A. chica chloroformic fraction (Ac-CF) obtained from leaves containing bioactive metabolites. The mutagenic effects were evaluated using the Salmonella mutagenicity assay, with TA98, TA97a, TA100, TA102, and TA1535 strains, with and without metabolic activation. In vivo mutagenic and genotoxic/antigenotoxic effects were investigated using the micronucleus (MN) test in bone marrow and alkaline comet assay in blood and liver after administration of 100, 500, or 1000 mg/kg Ac-CF in CF-1 mice by gavage (once a day for 3 d). In vitro antioxidant potential was evaluated using DPPH and xanthine/hypoxanthine assays. Ac-CF was not mutagenic in any of the Salmonella typhimurium strains tested and showed negative responses for mutagenicity and genotoxicity in mice. Further, Ac-CF displayed antigenotoxic effects by decreasing the oxidative DNA damage induced by hydrogen peroxide by greater than 50% in blood and liver. The antioxidant action detected in the in vitro assays demonstrated IC50 of 0.838 mg/ml in the xanthine/hypoxanthine assay and IC50 of 28.17 μg/ml in the DPPH assay. In conclusion, Ac-CF did not induce mutagenic and genotoxic effects and was able to protect DNA against oxidative damage in vivo, suggesting that this fraction may not pose genetic risks, although further toxicology assays are necessary. This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico); FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul); and GENOTOX-ROYAL, Brazil.

Micronucleus induction in the bone marrow of rats by pharmacological mechanisms. I: glucocorticoid receptor agonism

A novel selective glucocorticoid receptor (GR) agonist, AZD2906, was found to increase the incidence of micronucleated immature erythrocytes (MIE) in the bone marrow of rats given two oral doses at the maximum tolerated level. Because GR agonists as a class are considered not to be genotoxic and AZD2906 showed no activity in the standard in vitro tests or in vivo in a rat liver comet assay, investigative studies were performed to compare AZD2906 with a reference traditional GR agonist, prednisolone. Emphasis was placed on blood and bone marrow parameters in these studies because GR activation has been reported to induce erythropoiesis which, in turn, is known to increase MIE in the bone marrow. Both compounds induced almost identical, small increases in micronucleus frequency at all doses tested. Directly comparable changes in haematological and bone marrow parameters were also seen with significant decreases in lymphoid cells in both compartments and significant increases in numbers of circulating neutrophils. Although no evidence of increased erythropoiesis was seen as increased immature erythrocyte numbers either in the blood or in the bone marrow, histopathological examination showed focal areas in the bone marrow where the erythroid population was enriched in association with an atrophic myeloid lineage. This could have been due to direct stimulation of the erythroid lineage or a secondary effect of myelosuppression inducing a rebound increase in erythropoiesis into the vacant haematopoietic cell compartment. It was concluded that the increased MIE frequencies induced by both AZD2906 and prednisolone are a consequence of their pharmacological effects on the bone marrow, either by directly inducing erythropoiesis or by some other unknown effect on cellular function, and do not indicate potential genotoxicity. This conclusion is supported by the lack of carcinogenic risk in man demonstrated by decades of clinical use of prednisolone and other GR agonists.