Peripheral Blood Micronucleus Assay Research Articles

Toxicity Profile Study of Antihypertensive Drug Prazosin in Pregnant Wistar Rats

The purpose of this research is to investigate and assess the cytotoxicity and genotoxicity of Prazosin HCL in pregnant rats. Prazosin (PZ) was administered to the animals intraperitoneally (IP) at dosages of 5, 15, and 25 mg/kg/body weight for single dose (14-day) toxicity tests. The following parameters have been examined for evaluating genotoxicity: bone marrow micronucleus assay, peripheral blood micronucleus assay, DNA damage is measured using the metaphase chromosomal analysis, DNA damage is measured using the DNA fragmentation test, and cytotoxicity is measured using a histological analysis. The results obtained clearly demonstrate that PZ induced hazardous responses at the higher dose in the hepatocytes, as evidenced by DNA damage, and increased DNA fragmentation in pregnant rats. Observing that PZ significantly increased DNA strand breakage and structural chromosomal aberrations in bone marrow cell lines is also fascinating. As a result, it is thought to be genotoxic to bone marrow and mouse hepatocyte cells. The current study showed that Prazosin had significant genotoxic effects in pregnant rats at the matching hepatotoxic dose level.

Betulinic acid exerts antigenotoxic and anticarcinogenic activities via inhibition of COX-2 and PCNA in rodents.

Phytochemicals have been suggested as an effective strategy for cancer prevention. Within this context, triterpene betulinic acid (BA) exhibits several biological properties but its chemopreventive effect has not been fully demonstrated. The present study investigated the antigenotoxic potential of BA against doxorubicin (DXR)-induced genotoxicity using the mouse peripheral blood micronucleus assay, as well as its anticarcinogenic activity against 1,2dimethylhydrazine (DMH)-induced colorectal lesions in rats. Micronuclei (MN) assay and aberrant crypt foci assay were used to assess the antigenotoxic and the anticarcinogenic potential, respectively. The molecular mechanisms underlying the anticarcinogenic activity of BA were evaluated by assessing anti-inflammatory (COX-2) and antiproliferative (PCNA) pathways. The results demonstrated that BA at the dose of 0.5 mg/kg bodyweight exerted antigenotoxic effects against DXR, with a reduction of 70.2% in the frequencies of chromosomal damage. Animals treated with BA showed a 64% reduction in the number of preneoplastic lesions when compared to those treated with the carcinogen alone. The levels of COX-2 and PCNA expression in the colon were significantly lower in animals treated with BA and DMH compared to those treated with the carcinogen alone. The chemopreventive effect of BA is related, at least in part, to its antiproliferative and anti-inflammatory activity, indicating a promising potential of this triterpene in anticancer therapies, especially for colorectal cancer.

Combined markers for predicting cognitive deficit in patients with Alzheimer\u2019s disease

BackgroundAlzheimer’s disease (AD) is the most widely recognized type of dementia. It is associated with cell cycle abnormalities including genomic instability and increased micronuclei (MNi) which usually evolve many years before the appearance of the clinical manifestations. Digital electroencephalogram (EEG) has a role in perceiving brain changes in dementia and in early detection of cognitive decline. This study aimed to assess the competency of using neurophysiological markers including absolute power of alpha waves and a cytogenetic marker which comprises scoring of MNi as a step toward early and preclinical diagnosis of AD. The study was conducted on 27 subjects; they were 15 patients diagnosed as sporadic AD and a group of 12 age and sex-matched controls. All subjects were subjected to Mini-Mental State Examination (MMSE), conventional EEG, digital EEG, and cytokinesis-block micronucleus assay (CBMN) in peripheral blood lymphocytes.ResultsConventional EEG showed a normal background activity with no abnormal epileptogenic discharges in both groups. Digital EEG showed significant reduction of the absolute power of alpha waves for AD patients as compared to the control group (P < 0.0001). Score of MNi showed statistical significant difference between the two groups (P < 0.0001). By linking scores of both cognitive state using MMSE and MNi among the group of patients, a significant negative correlation was detected (r = −0.6066). The correlations between cognitive state and the absolute power of alpha wave among the patients revealed a positive correlation (r = 0.2235).ConclusionsThe combination of both cytogenetic and neurophysiological markers can be beneficial for early detection of cognitive decline and may lead to preclinical identification of individuals at increased risk for AD, where at this stage treatment is constructive. The negative correlation between the scores of MNi and MMSE is suggestive for the impact of genomic instability on the cognitive state.

Assessment of DNA damage through micronucleus studies in subjects exposed to formalin

Background: Formaldehyde (FA) is the reactive and simplest of all the aldehydes. It is used as a preservative in anatomy, pathology and forensic laboratories. The international agency for research on cancer has classified FA as a carcinogen that can cause nasopharyngeal carcinoma, Leukaemia, Liver and pancreatic cancer. Objective And Method: The aim of the study was to assess the DNA damage in peripheral blood lymphocytes and in buccal cells by Micronucleus assay in Formalin exposed workers of Anatomy, Pathology and Forensic laboratories and compare with the control group, and also to analyze the relationship between frequency of Micronuclei and duration of exposure to formalin. Results: The mean and standard deviation (SD) of micronuclei in peripheral blood of exposed was 8.35 and in controls was 4.18. There was a significant increase in the frequency of MN in exposed group when compared with the comparison group (p&lt;0. 5876). Pearson’s correlation test showed a positive correlation between the years of FA exposure and the number of micronuclei in buccal cells and peripheral blood indicating that DNA damage due to FA was directly proportional to the duration of exposure (r=0.8, 0.9). Conclusion: The present study was done to assess the DNA damage in people who were exposed to FA and a control group not exposed to FA by buccal cell and peripheral blood Micronucleus Assay. There was a significant increase in the MN in people exposed to FA which was directly proportional to the duration of exposure.

In vitro and in vivo effects of 5-aminotetrazole (5-AT), an energetic compound

Perchlorate is an important oxidizer used in propellants, pyrotechnics, and as a gas generator in commercial airbags, fireworks, and roadside flares. It is highly water soluble, interferes with thyroidal iodide uptake and is an environmental contaminant. By changing the reaction chemistry, 5-aminotetrazole (5-AT) and nitrates replace perchlorate in some propellants. The short term toxicity of 5-AT was evaluated. Using a modified Ames assay, 5-AT was not mutagenic with or without S9 metabolic activation. 5-AT was considered “slightly toxic” with an EC50 of 28.8 mg 5-AT/L for a 15 min exposure in Aliivibrio fischeri. In the in vitro sodium iodide symporter test, 5-AT did not inhibit the uptake of iodine. In the acute rat oral test, no adverse effects and no mortalities were observed at the limit dose of 2000 mg 5-AT/kg. In the 14-day sub-acute study, there were no clinical signs of toxicity or morbidity up to 623 mg 5-AT/kg-day; the highest dose tested. No differences were observed in hematology, clinical chemistry, organ weight, body weight, food consumption, histopathology, or DNA damage (peripheral blood micronucleus assay) of treatments compared with controls. The No Observed Adverse Effect Level (NOAEL) was 623 mg 5-AT/kg-day, the highest dose in the subacute oral bioassay.

Integration of micronucleus, comet, and Pig-a gene mutation endpoints into rat 15-day repeat-treatment studies: Proof-of-principle with Auramine O

A series of genotoxicity assessments were conducted on male Sprague Dawley rats treated with Auramine O (AO) to establish a multiple-endpoint assay. The rat liver micronucleus assay, in combination with the comet assay, peripheral blood micronucleus assay, and erythrocyte Pig-a assay in the same experiment, comprehensively assess the genotoxicity of AO. Rats were orally exposed to 0, 100, 200, or 400 mg/kg/day AO for 15 consecutive days. The blood was sampled on Days -1 and 15 for the erythrocyte Pig-a assay and peripheral blood micronucleus assay. Livers were sampled on Day 15 for the liver micronucleus assay and comet assay. Based on the liver micronucleus assay and liver comet assay, AO induced a significant dose-related increase of micronucleated hepatocyte frequencies, and tail DNA percentages, respectively in the middle- and high-dose groups. On the blood micronucleus test and Pig-a assay, no significant increases were observed for the micronucleated reticulocyte frequencies, mutant erythrocyte frequencies (RBCCD59−) or mutant reticulocyte frequencies (RETCD59−) at any of the time points studied. In conclusion, using a multiple-endpoint genotoxicity assay method can reduce the number of experimental animals, boost the efficiency of the experiment, and improve the accuracy of investigations of genotoxicity.

SB-83, a 2-Amino-thiophene derivative orally bioavailable candidate for the leishmaniasis treatment

Leishmaniasis, affecting more than 12 million people worldwide has become a severe public-health problem. The therapeutic arsenal against leishmaniasis is mainly administered by parenteral route; it is toxic, expensive, and associated with recurrence risk. The need for further therapeutic compounds research is pressing. In previous studies, we demonstrated the antileishmanial activities of ten 2-amino-thiophene derivatives, which evidenced the action of a compound, called SB-83, having expressive antileishmania activity in an in vitro infection model. In the present work, we describe preclinical studies of the thiophenic derivative SB-83, such as acute toxicity, genotoxicity, in vivo oral efficacy in a murine model, and in vitro antileishmanial activity against an L. amazonensis SbIII-resistant strain. Determining acute preclinical toxicity, the LD50 of SB-83 was estimated at 2500 mg/kg orally, with few behavioral changes in Swiss mice. Further, treatment with 2000 mg/kg of SB-83 did not induce in vivo genotoxic activity in the peripheral blood micronucleus assay. In 7 weeks of oral treatment, SB-83 reduced paw lesion size in L. amazonensis infected mice by 52.47 ± 5.32%, and decreased the parasite load of the popliteal lymph node and spleen at the highest dose tested (200 mg/kg) respectively by 42.57 ± 3.14%, and 100%, without presenting weight change or other changes of clinical importance in the biochemical and hematological profiles. The treatment of promastigotes and intracellular amastigotes of SbIII sensitive and resistant strains with SB-83 did not produce differences in antileishmania activity, which suggests no cross-resistance. Thus, this work demonstrated that SB-83 has potential as a new active drug candidate even when orally administered, which may become a new therapeutic alternative for the treatment of leishmaniasis.

Genotoxic and cytotoxic evaluation of Jatropha dioica Sessé ex Cerv. by the micronucleus test in mouse peripheral blood

Jatropha dioica Sessé ex Cerv. is a medicinal plant credited with low cytotoxicity in vitro. Thus, the objective of this work was to evaluate the possible genotoxic and cytotoxic effect in vivo of the J. dioica aqueous extract by means of micronucleus assay in mouse peripheral blood. Four different J. dioica aqueous extract dose-units were evaluated (30, 60, 100, and 300 mg/kg). The extract was administered orally to male Balb-C-strain mice every 24 h during 5 days. Blood samples were taken at 0, 24, 48, 72, 96, and 120 h from the mouse's tail and were performed in duplicate extensions. The number of Polychromatic Erythrocytes (PCE), Polychromatic Micronucleus Erythrocytes (PCEMN), and Micronucleus Erythrocytes (MNE) was determined at the different sampling times in the different study groups. Our results showed that the group that received 60 mg/kg of cyclophosphamide (positive control) presented a significant decrease in the PCE (p = 0.044) proportion and a significant increase in MNE (p = 0.032, p = 0.0001). The groups that received the different J. dioica aqueous extract doses did not present either a PCE decrease or an increase in PCEMN and MNE. J. dioica exerts neither a genotoxic nor a cytotoxic effect on mouse peripheral blood at high doses.

Effectiveness of the liver micronucleus assay using juvenile mice.

This study investigated the effectiveness of the liver micronucleus (MN) assay using juvenile mice. Therefore, we analyzed various hepatic cytochrome P450 (CYP)- mediated activities of ethoxyresorufin O-deethylation, pentoxyresorufin O-dealkylation, tolbutamide hydroxylation, bufuralol 1’-hydroxylation, aniline hydroxylation and midazolam 4-hydroxylation by CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A, respectively, in non-treated male ICR mice aged between 3 and 8 weeks. The enzyme efficiency levels in 3- and 4-week-old mice were approximately similar to or higher than those in 8-week-old mice, except for CYP1A and CYP2E in 3- and 4-week-old mice, respectively. Since these results suggest that juvenile mice have sufficient activities for most CYP enzymes, we also conducted a liver MN assay using diethylnitrosamine (DEN), a rodent hepatocarcinogen, on male ICR mice aged between 3 and 6 weeks. A peripheral blood (PB) MN assay was performed simultaneously in 4-week-old mice. Assays incorporating DEN produced positive results in 3- and 4-week-old mice and showed a dose-dependent increase in the micronucleated hepatocyte frequencies at 4 weeks. Both the liver MN assay in 5- and 6-week-old mice and the PB MN assay had negative results when using DEN. These results suggest that 3- and 4-week-old mice have micronuclei-inducing potential in the liver to detect genotoxic compounds using the liver MN assay.

Antimutagenic Effects of Selenium-Enriched Polysaccharides from Pyracantha fortuneana through Suppression of Cytochrome P450 1A Subfamily in the Mouse Liver.

Both selenium (Se) and polysaccharides from Pyracantha fortuneana (Maxim.) Li (PFPs) (P. fortuneana) have been reported to possess antioxidative and immuno-protective activities. Whether or not Se-containing polysaccharides (Se-PFPs) have synergistic effect of Se and polysaccharides on enhancing the antioxidant and immune activities remains to be determined. We previously reported that polysaccharides isolated from Se-enriched P. fortuneana (Se-PFPs) possessed hepatoprotective effects. However, it is not clear whether or not they have anti-mutagenic effects. In the present study, we compared and evaluated anti-mutagenic effects of Se-PFPs at three concentrations (1.35, 2.7 and 5.4 g/kg body weight) with those of PFPs, Se alone or Se + PFPs in mice using micronucleus assay in bone marrow and peripheral blood as well as mitomycin C-induced chromosomal aberrations in mouse testicular cells. We also elucidated the underlying mechanism. Our results demonstrated that Se-PFPs inhibited cyclophosphamide (CP)-induced micronucleus formation in both bone marrow and peripheral blood, enhanced the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in mouse liver, and reduced the activity and expression of cytochrome P450 1A (CYP4501A) in mouse liver in a dose-dependent manner. In addition, we found that the anti-mutagenic potential of Se-PFPs was higher than those of PFPs, Se alone or Se + PFPs at the same level. These results suggest that the anti-mutagenic potential of Se-PFPs may be mediated through the inhibition of the activity and expression of CYP4501A. This study indicates that application of Se-PFPs may provide an alternative strategy for cancer therapy by targeting CYP1A family.

Evaluation of the repeated-dose liver, bone marrow and peripheral blood micronucleus and comet assays using kojic acid

The repeated-dose liver micronucleus assay has the potential to detect liver carcinogens and could be integrated into general toxicological studies. To assess the performance of this assay, kojic acid was tested in 14-day and 28-day liver micronucleus assays. We evaluated the incidence of micronucleated cells in liver, bone marrow and peripheral blood and performed comet assays in both the liver and peripheral blood (comet assay was performed only for 14-days). Kojic acid, a skin-whitening agent used in cosmetic products, was orally dosed in six-week-old male rats at 250, 500 and 1000mg/kg/day for 14 days, and at 125, 250 and 500mg/kg/day for 28 days. Organ weight and histopathology were examined at the end of the experiment. Neither a clear, positive response in micronucleus (MN) incidence nor changes in the percent of tail DNA in the comet assays was noted in liver and bone marrow. An increase of relative liver weight was observed in 1000mg/kg/day for 14 days. In histopathology, minimal hypertrophy of hepatocytes was found at 1000mg/kg/day for 14 days. The results of both the micronucleus assay and the comet assay indicate that 14-day and 28-day repeated dosing of kojic acid are non-genotoxic in the liver and bone marrow.Kojic acid has been known to act as a tumor-promoter in thyroid carcinogenesis but has not been shown to have initiation activities in liver carcinogenesis. Findings in this study are consistent with the evidence that kojic acid is not an apparent initiator of liver carcinogenesis. Therefore, the liver micronucleus assay is simple and sensitive to detect genotoxic liver carcinogens.

3-Aminobenzamide – a PARP inhibitor enhances the sensitivity of peripheral blood micronucleus and comet assays in mice

Context: DNA repair is an essential outcome of DNA damage, which may compromise the end point of various in vitro and in vivo test systems of the genotoxicity evaluation. poly(ADP-ribose) polymerase (PARP) enzymes have an essential role in DNA repair. Here, we investigated the effect of 3-AB, a PARP inhibitor on the sensitivity of comet and PBMN assays.Objective: This study aimed to enhance the sensitivity of the comet and peripheral blood micronucleus (PBMN) assays using 3-aminobenzamide (3-AB), a well-characterized PARP inhibitor.Materials and methods: Cyclophosphamide (CP, 50 mg/kg), 5-flourouracil (5-FU, 25 mg/kg), zidovudine (AZT, 400 mg/kg) and furosemide (FUR, 60 mg/kg) were selected as genotoxins. 3-AB was given every 8 h with the first dose given 2 h before the genotoxin treatment. For the PBMN assay, small amount of blood was taken from the tail tip of each animal and smears were prepared. The comet assay was performed in PBL, bone marrow and liver.Results: In the comet as well as PBMN assay, 3-AB pre-treatment enhanced the extent of DNA damage in all the combination groups (3-AB + CP, 3-AB + 5-FU and 3-AB + AZT) compared to CP, 5-FU and AZT per se. 3-AB also enhanced the DNA damage caused by FUR in the bone marrow and liver.Discussion: This study results clearly demonstrate that the pretreatment with 3-AB (30 mg/kg) significantly enhances the sensitivity of the PBMN and comet assays. This model may be useful for the detection of marginally active DNA damaging agents.

Flow cytometric analysis of micronuclei in rat peripheral blood: An interlaboratory reproducibility study

In anticipation of proposed OECD guideline changes that may include increasing the number of reticulocytes scored for micronuclei, an inter-laboratory reproducibility study of the rat peripheral blood micronucleus assay was performed using flow cytometry. In this experiment, male Sprague–Dawley (SD) rats were treated with the model clastogen cyclophosphamide (CP: 5, 10 or 15mg/kg) by a single oral administration. As controls, rats were treated with physiological saline (solvent) in the same manner as for the model clastogen. Peripheral blood was collected from each rat 48h after the treatment. The blood samples were prepared at the Public Interest Incorporated Foundation, BioSafety Research Center (BSRC) in duplicate using the rat MicroFlowPLUS Kit. After fixation, one replicate set of samples was shipped to Litron Laboratories, and each sample was analyzed by flow cytometry at the two laboratories. In addition, the frequency of micronucleated reticulocytes (MNRETs) was determined at the BSRC by microscopic analysis using supravital acridine orange (AO) staining. The reproducibility of micronucleated reticulocyte frequencies analyzed by microscopy and flow cytometry showed good correlation (r2=0.84). The frequencies of micronucleated reticulocytes analyzed by flow cytometry at the two independent laboratories showed good concordance (r2=0.97). The data indicate that the flow cytometric micronucleus analysis method is a good alternative to manual microscopic analysis. Flow cytometry allows groups to readily score 5000 or even 20,000 RETs in a matter of minutes compared to manual analysis. This results in increased reliability of the assay by achieving better statistical power.

Repeated dose liver micronucleus assay using adult mice with multiple genotoxicity assays concurrently performed as a combination test.

Recently, the liver micronucleus (MN) assay using young adult rats with repeated administrations has been investigated by employing a new method without partial hepatectomy orin situcollagenase perfusion as the repeated dose liver MN (RDLMN) assay by Narumiet al. (2012). In our study, in order to investigate the possibility of the RDLMN assay using young adult mice instead of rats and the feasibility of employing some genotoxicity assays along with the RDLMN assay as a combination test, two genotoxic carcinogens (N,N-diethylnitrosoamine (DEN) and cisplatin (CIS)) and a nongenotoxic carcinogen (phenobarbital sodium (PHE)) were administered to mice for 15 or 29 days. Then, the liver MN assay, peripheral blood (PB) MN assay and comet assay using the liver and kidney were concurrently performed as a combination test. DEN showed positive responses to all endpoints except MN induction in PB after 15 days of repeat administration. A cross-linking agent, CIS, showed MN induction in liver after 29 days of repeat administration, and in PB after 15 and 29 days of repeat administration, although the comet assay yielded negative responses for both organs at both sampling times. PHE yielded negative responses for all endpoints. In conclusion, it is suggested that the RDLMN assay using mice is a feasible method to be integrated into the general repeated toxicity test along with the combination assays, i.e., comet assay or PB MN assay, which would help in risk assessment for carcinogenicity by comparing the results of combination assays with each other.

The synthetic food colouring agent Allura Red AC (E129) is not genotoxic in a flow cytometry-based micronucleus assay in vivo

The safety of several azo colouring agents, used as food additives, has during the years been questioned. Allura Red AC (E129) has in some publications been classified as genotoxic. In fact, in the European Union, Allura Red is permitted as a food additive in human food, but, surprisingly, it was not acceptable as an additive for use in animal feed. In this study we have evaluated whether Allura Red is genotoxic using a flow cytometer-based micronucleus assay in peripheral blood of mice. Male FVB mice were given a single intra-peritoneal injection of various doses of Allura Red and sacrificed at 46 h after treatment. The tested doses were 0, 100, 200, 400, 600, 800, 1000, 1500, and 2000 mg/kg body weight (b.w.). Each dose group constituted three mice, except for in the dose group of 1000 mg/kg b. w., which constituted four mice. Blood samples were collected and the frequency of micronucleated polychromatic erythrocytes (fMNPCE) and the cell proliferation (%PCE) was determined. The analyses did not show any significant difference in the %PCE or in the fMNPCE. Consequently, under the testing circumstances one can conclude that Allura Red is not genotoxic.

Pretreatment with valproic acid, a histone deacetylase inhibitor, enhances the sensitivity of the peripheral blood micronucleus assay in rodents

Micronucleus (MN) assay is widely used for the determination of the genotoxic potential of new chemical entities. Improvement in the sensitivity of MN assay will be advantageous for the successful detection of marginally active genotoxins. In the past, several improvements have been made in the automated scoring of micronuclei, while very few attempts have been taken to improve the sensitivity of manual micronuclei detection. The present study aims to validate the effect of valproic acid (VPA) pretreatment on the sensitivity of peripheral blood micronucleus (PBMN) assay using cyclophosphamide (CP, 50mg/kg), methotrexate (MTX, 20mg/kg) and zidovudine (AZT, 400mg/kg) in rodents. However, to find out the optimum VPA pretreatment time as well as to detect the effect of species and age difference, separate experiments were conducted on young Swiss albino mice (24–28 days) and Sprague-Dawley rats (21–24 days), in which significant increase in MN induction was observed with 3-day VPA pretreatment in both the species. Based on these results, studies on adult mice were conducted with 3-day VPA pretreatment along with CP or MTX or AZT. The results of the present study clearly demonstrate that the 3-day VPA pretreatment significantly enhances the sensitivity of PBMN assay in peripheral blood (PB) in adult mice. After validation with other standard genotoxins as well as other HDAC (histone deacetylase) inhibitors, this model may be useful for the detection of marginally active DNA damaging agents.

Comparison in the efficiency of different murine lines for genotoxicity assays.

The aim of this research was to compare the efficiency of different murine lines for genotoxicity assays. Rats and mice of different murine lines were used. The spontaneous and induced indexes were evaluated according to alkaline comet assay of peripheral blood leukocytes, micronucleus and chromosomic aberration assay of bone marrow cell, and sperm head morphology assay. In most of the evaluated assays the line of Balb/c mice turned out to be the ideal biomodel, with less spontaneous indexes and high induced indexes to the mutagen used; allowing to detect in a narrow error margin those substances that are classified of very low genotoxicity. These results demonstrate that genetically the line of Balb/c mice in both sexes is more stable than the other ones evaluated. This suggests the use of the Balb/c line on in vivo genotoxicity assay will increase sensibility and robustness.

Investigation on the protective effects of cranberry against the DNA damage induced by benzo[a]pyrene.

There are few reports that demonstrate the antigenotoxic potential of cranberries. Although the types of berry fruits consumed worldwide are many, this paper focuses on cranberries that are commonly consumed in Mexico (Vaccinium macrocarpon species). The purpose of the present study is to determine whether cranberry ethanolic extract (CEE) can prevent the DNA damage produced by benzo[a]pyrene (B[a]P) using an in vivo mouse peripheral blood micronucleus assay. The experimental groups were organized as follows: a negative control group (without treatment), a positive group treated with B[a]P (200 mg/kg), a group administered with 800 mg/kg of CEE, and three groups treated with B[a]P and CEE (200, 400, and 800 mg/kg) respectively. The CEE and benzo[a]pyrene were administered orally for a week, on a daily basis. During this period the body weight, the feed intake, and the determination of antigenotoxic potential were quantified. At the end of this period, we continued with the same determinations for one week more (recovery period) but anymore administration of the substances. The animals treated with B[a]P showed a weight increase after the first week of administration. The same phenomenon was observed in the lots combined with B[a]P and CEE (low and medium doses). The dose of 800 mg/kg of CEE showed similar values to the control group at the end of the treatment period. In the second part of the assay, when the substances were not administered, these experimental groups regained their normal weight. The dose of CEE (800 mg/kg) was not genotoxic nor cytotoxic. On the contrary, the B[a]P increases the frequency of micronucleated normochromatic erythrocytes (MNNE) and reduces the rate of polychromatic erythrocytes (PE) at the end of the treatment period. With respect to the combined lots, a significant decrease in the MN rate was observed from the sixth to the eighth day of treatment with the two high doses applied; the highest protection (60%) was obtained with 800 mg/kg of CEE. The same dose showed an anticytotoxic effect which corresponded to an improvement of 62.5% in relation to the animals administered with the B[a]P. In the second period, all groups reached values that have been seen in the control group animals. Our results suggest that the inhibition of clastogenicity of the cranberry ethanolic extract against B[a]P is related to the antioxidant capacity of the combination of phytochemicals present in its chemical composition.

In vivo genotoxicity of furan in F344 rats at cancer bioassay doses

Furan, a potent rodent liver carcinogen, is found in many cooked food items and thus represents a human cancer risk. Mechanisms for furan carcinogenicity were investigated in male F344 rats using the in vivo Comet and micronucleus assays, combined with analysis of histopathological and gene expression changes. In addition, formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIII)-sensitive DNA damage was monitored as a measure of oxidative DNA damage. Rats were treated by gavage on four consecutive days with 2, 4, and 8mg/kg bw furan, doses that were tumorigenic in 2-year cancer bioassays, and with two higher doses, 12 and 16mg/kg. Rats were killed 3h after the last dose, a time established as producing maximum levels of DNA damage in livers of furan-treated rats. Liver Comet assays indicated that both DNA strand breaks and oxidized purines and pyrimidines increased in a near-linear dose-responsive fashion, with statistically significant increases detected at cancer bioassay doses. No DNA damage was detected in bone marrow, a non-target tissue for cancer, and peripheral blood micronucleus assays were negative. Histopathological evaluation of liver from furan-exposed animals produced evidence of inflammation, single-cell necrosis, apoptosis, and cell proliferation. In addition, genes related to apoptosis, cell-cycle checkpoints, and DNA-repair were expressed at a slightly lower level in the furan-treated livers. Although a mixed mode of action involving direct DNA binding cannot be ruled out, the data suggest that furan induces cancer in rat livers mainly through a secondary genotoxic mechanism involving oxidative stress, accompanied by inflammation, cell proliferation, and toxicity.

Lack of Mutagenicity of SMD-502, a New Vitamin D3 Analog for Topical Application, in Skin and Liver of gpt delta Transgenic Mice and in GDL1 Cells

SMD-502, a new vitamin D3 (VD3) analog, is an antipsoriatic drug candidate. The compound exhibited fewer side effects than known VD3 analogs in animal models, probably because the compound is rapidly converted into pharmacologically inactive form after permeating into systemic circulation from the application site on skin. This feature of the compound makes it difficult to assess genotoxic risk in vivo with the standard approach of bone marrow or peripheral blood micronucleus assays in rodents because of the low blood concentration level. To evaluate in vivo mutagenicity of the compound in the present study, mutant frequency (MF) in skin and liver of gpt delta transgenic mice was examined with percutaneous administration of SMD-502 for 28 days. In tissues collected 7 days after the end of administration, no significant increase in the MF was observed in either skin or liver. Additionally, when the compound was tested in a GDL1 cell line established from gpt delta mice, GDL1 cells exhibited no significant increase in MFs even under conditions in which they would be exposed to a much higher concentration of the compound than in the in vivo study. The results in this study further supported the consideration that SMD-502 has no mutagenic activity.