Assessment of imidacloprid-induced mutagenic effects in somatic cells of Swiss albino male mice.

Pesticides are being used for plant protection to increase food protection and to reduce insect-borne diseases worldwide. Exposure to the pesticides may cause genotoxic effects on both the target and nontarget organisms, including man. Therefore, the mutagenicity evaluation of such pesticides has become a priority area of research. Imidacloprid (IMI), a neonicotinoid insecticide, is widely used in agriculture either alone or in combination with other insecticides. A combined approach employing micronucleus test (MNT) and chromosomal aberrations assay (CA) was utilized to assess the mutagenicity of imidacloprid in bone marrow of Swiss albino male mice. IMI suspension was prepared in 3% gum acacia and administered at doses of 5.5, 11 and 22 mg/kg body weight for 7, 14 and 28 days to mice. IMI treatment resulted in a dose and time-dependant increase in the frequencies of micronuclei per cell and chromosomal aberrations in bone marrow cells. A statistically significant increase in chromosomal aberrations and micronuclei/cell was found only after daily treatment of IMI at highest selected dose (22 mg/kg body weight) for longest selected time period (28 days) compared to the control group. Thus, daily exposure of imidacloprid at a dose level of 22 mg/kg body weight for 28 days caused mutagenic effects on the somatic cells of Swiss albino male mice.

Automation of the in vitro micronucleus and chromosome aberration assay for the assessment of the genotoxicity of the particulate and gas-vapor phase of cigarette smoke.

Total particulate matter (TPM) and the gas-vapor phase (GVP) of mainstream smoke from the Reference Cigarette 3R4F were assayed in the cytokinesis-block in vitro micronucleus (MN) assay and the in vitro chromosome aberration (CA) assay, both using V79-4 Chinese hamster lung fibroblasts exposed for up to 24 h. The Metafer image analysis platform was adapted resulting in a fully automated evaluation system of the MN assay for the detection, identification and reporting of cells with micronuclei together with the determination of the cytokinesis-block proliferation index (CBPI) to quantify the treatment-related cytotoxicity. In the CA assay, the same platform was used to identify, map and retrieve metaphases for a subsequent CA evaluation by a trained evaluator. In both the assays, TPM and GVP provoked a significant genotoxic effect: up to 6-fold more micronucleated target cells than in the negative control and up to 10-fold increases in aberrant metaphases. Data variability was lower in the automated version of the MN assay than in the non-automated. It can be estimated that two test substances that differ in their genotoxicity by approximately 30% can statistically be distinguished in the automated MN and CA assays. Time savings, based on man hours, due to the automation were approximately 70% in the MN and 25% in the CA assays. The turn-around time of the evaluation phase could be shortened by 35 and 50%, respectively. Although only cigarette smoke-derived test material has been applied, the technical improvements should be of value for other test substances.

Forskolin: genotoxicity assessment in Allium cepa.

Forskolin, a diterpene, 7ß-acetoxy-8,13-epoxy-1α,6ß,9α-trihydroxy-labd-14-en-11-one (C22H34O7) isolated from Coleus forskohlii, exerts multiple physiological effects by stimulating the enzyme adenylate cyclase and increasing cyclic adenosine monophosphate (cAMP) concentrations. Forskolin is used in the treatment of hypertension, congestive heart failure, eczema, and other diseases. A cytogenetic assay was performed in Allium cepa to assess possible genotoxic effects of forskolin. Forskolin was tested at concentrations 5-100 µM for exposure periods of 24 or 48 h. Treated samples showed significant reductions in mitotic index (p < 0.05) and increases in the frequency of chromosome aberrations (p < 0.01) at both exposure times. The treated meristems showed chromosome aberrations including sticky metaphases, sticky anaphases, laggard, anaphase bridges, micronuclei, polyploidy, fragments, breaks, and C-mitosis. Forskolin may cause genotoxic effects and further toxicological evaluations should be conducted to ensure its safety.

Suitability of scoring PCC rings and fragments for dose assessment after high-dose exposures to ionizing radiation.

Assessment of radiation doses through measurement of dicentric chromosomes may be difficult due to the inability of damaged cells to reach mitosis. After high-dose exposures, premature chromosome condensation (PCC) has become an important method in biodosimetry. PCC can be induced upon fusion with mitotic cells, or by treatment with chemicals such as calyculin A or okadaic acid. Several different cytogenetic endpoints have been measured with chemically induced PCC, e.g., via scoring of extra chromosome pieces or ring chromosomes. The dose-effect curves published with chemically induced PCC show differences in their coefficients and in the distribution of rings among cells. Here we present a study with calyculin A to induce PCC in peripheral blood lymphocytes irradiated at nine different doses of γ-rays up to 20Gy. Colcemid was also added in order to observe metaphase cells. During microscopical analysis the chromosome aberrations observed in the different cell-cycle phases (G2/M-PCC, M/A-PCC and M cells) were recorded. The proportion of G2/M-PCC cells was predominant from 3 to 20Gy, M cells decreased above 1Gy and M/A-PCC cells remained constant at all doses and showed the highest frequencies of PCC rings. Depending on the cell-cycle phase there was a difference in the linear coefficients in the dose-effect curves of extra fragments and rings. Poisson distribution among PCC rings was observed after calyculin A+colcemid treatment, facilitating the use of this methodology also for partial body exposures to high doses. This has been tested with two simulated partial exposures to 6 and 12Gy. The estimated doses in the irradiated fraction were very close to the real dose, indicating the usefulness of this methodology.

Assessment of cytotoxic and clastogenic effects of nimesulide: an NSAID drug in somatic cells of BALB/c mice in vivo.

The in vivo genotoxicity of nimesulide, a sulfononilide nonsteroidal anti-inflammatory drug (NSAID) with anti-inflammatory, analgesic, and antipyretic effects, was evaluated by employing a mouse in vivo chromosomal aberration test in bone marrow cells. Oral treatment of animals for 5 consecutive days with 1, 2.5, 5, and 7.5 mg/kg body weight of the drug resulted in a statistically nonsignificant reduction in mitotic index and increase in CAs/cell and percent abnormal metaphase. The results indicated that nimesulide does not induce cytotoxicity and is a weak clastogen in the bone marrow cells of the mouse in vivo. Thus, the drug presents a very weak genotoxic risk.

Genetic toxicology assessment of HI-6 dichloride.

The oxime HI-6 dichloride [1-(2 hydroxyiminomethyl -1-pyridino)-3-(4-carbamoyl-1-pyridino)-2-oxapropane dichloride monohydrate] has shown to be a potent reactivator of cholinesterase activity and may have efficacy for the treatment of organophosphate intoxication [SIPRI, 1976; Schenk et al.; Arch Toxicol 36:71-81, 1976]. As part of a preclinical safety assessment program, the genetic toxicology of HI-6 dichloride was evaluated in a series of assays designed to measure induction of gene mutations and chromosomal aberrations. HI-6 dichloride gave negative responses in the Salmonella mutagenicity assay and in the CHO/HGPRT gene mutation assay. Dose-dependent increases in the frequency of chromosomal aberrations were noted when HI-6 dichloride was tested in cultured CHO cells and in cultured human peripheral blood lymphocytes. The mouse lymphoma gene mutation assay, reputed to measure both gene mutations and chromosomal deletions, was negative in the absence of metabolic activation. Depending on the criteria employed, a negative or equivocal response was seen in the presence of rat liver-derived S-9 mix. An in vivo rat bone marrow metaphase assay performed to further investigate the in vitro clastogenic responses was negative. The results from these studies indicate that HI-6 dichloride does not induce gene mutations in vitro; however, it is clastogenic in vitro but does not appear to be clastogenic in vivo.