Environmental genotoxicity evaluation using cytogenetic end points in wild rodents

We analyzed cytogenetic end points in three populations of two species of wild rodents--Akodon montensis and Oryzomys nigripes--living in an industrial, an agricultural, and a preservation area at the Itajaí Valley, state of Santa Catarina, Brazil. Our purpose was to evaluate the performance of the following end points in the establishment of a genotoxic profile of each area: the polychromatic/normochromatic cell ratio; the mitotic index; the frequency of micronucleated cells both in the bone marrow and peripheral blood; and the frequency of cells with chromosome aberrations in the bone marrow. Preparations were obtained using conventional cytogenetic techniques. The results showed a) the role of the end points used as biomarkers in the early detection of genotoxic agents and in the identification of species and populations at higher risk; b) the difference in sensitivity of the species selected as bioindicators in relation to the cytogenetic end points analyzed; c) the need to use at least two sympatric species to detect the presence of genotoxins in each locality; and d) the need to use several end points when trying to establish a genotoxic profile of an area.     

Exploring the molecular mechanisms of nickel-induced genotoxicity and carcinogenicity: a literature review

Nickel, a naturally occurring element that exists in various mineral forms, is mainly found in soil and sediment, and its mobilization is influenced by the physicochemical properties of the soil. Industrial sources of nickel include metallurgical processes such as electroplating, alloy production, stainless steel, and nickel-cadmium batteries. Nickel industries, oil- and coal-burning power plants, and trash incinerators have been implicated in its release into the environment. In humans, nickel toxicity is influenced by the route of exposure, dose, and solubility of the nickel compound. Lung inhalation is the major route of exposure for nickel-induced toxicity. Nickel can also be ingested or absorbed through the skin. The primary target organs are the kidneys and lungs. Other organs such as the liver, spleen, heart, and testes can also be affected to a lesser extent. Although the most common health effect is an allergic reaction, research has also demonstrated that nickel is carcinogenic to humans. The focus of the present review is on recent research concerning the molecular mechanisms of nickel-induced genotoxicity and carcinogenicity. We first present a background on the occurrence of nickel in the environment, human exposure, and human health effects.     

New poliovirus vaccines: a molecular approach

This article summarizes recent work on the determinants of antigenicity in poliovirus type 3 and reports on experiments in progress aimed at understanding the molecular basis of attenuation in Sabin's type 3 vaccines. Ways in which this new information might be used to produce alternative, safe, inexpensive, multivalent vaccines against polio and other enteroviruses are discussed.     

Toxicological assessment of industrial solvents using human cell bioassays: assessment of short-term cytotoxicity and long-term genotoxicity potential

There is an increasing demand for simple toxicological screening methods to assess the human health risk associated with exposure to environmental toxicants. Such screening tools should allow for risk evaluation in terms of both short-term/acute toxicity and longer-term genetic damage, which may lead to mutagenicity and carcinogenicity. We employed a battery of human cell bioassays using the human hepatoma cell-line, HepG2, to assess the cytotoxic and genotoxic potential of environmental pollutants. Here, we demonstrate direct application of these human cell bioassays to the toxicological assessment of a number of industrial solvents that are in common use worldwide. HepG2 cells were exposed to various solvents at concentrations ranging from 25 to 500 ppm. The cells were then analysed using specific protocols for four different adverse effects: cell death/acute cytotoxicity using a neutral red uptake assay, altered enzyme function (often an indicator of cell stress) using the ethoxyresorufin O-deethylase (EROD) bioassay, DNA single strand breaks (SSB), and DNA repair induction, which evaluates mutagenic activity. Using the positive controls, linear dose-response curves were achieved for all four bioassays. The high sensitivity of the tests allowed for environmentally meaningful assessments, and precision studies showed excellent reproducibility for all four bioassays. Comparing the results of the four bioassays on each of 16 industrial solvents allowed for ranking of the anticipated relative human toxicity of these solvents, which were comparable with data from standard toxicity tests and human occupational data. Overall, the study clearly supports the application of the HepG2 cell bioassay system for rapid toxicological screening of many candidate toxicants for both short- and long-term toxicity potential.     

Assessment of hormonal activities and genotoxicity of industrial effluents using in vitro bioassays combined with chemical analysis

Wastewaters from various industries are a main source of the contaminants in aquatic environments. The authors evaluated the hormonal activities (estrogenic/anti-estrogenic activities, androgenic/anti-androgenic activities) and genotoxicity of various effluents from textile and dyeing plants, electronic and electroplate factories, pulp and paper mills, fine chemical factories, and municipal wastewater treatment plants in the Pearl River Delta region by using in vitro bioassays (yeast estrogen screen [YES]; yeast androgen screen [YAS]; and genotoxicity assay [umu/SOS]) combined with chemical analysis. The results demonstrated the presence of estrogenic, anti-estrogenic, and anti-androgenic activity in most industrial effluents, whereas no androgenic activities were detected in all of the effluents. The measured estrogenic activities expressed as estradiol equivalent concentrations (EEQs) ranged from below detection (3 of 26 samples) to 40.7 ng/L, with a mean of 7.33 ng/L in all effluents. A good linear relationship was found between the EEQs measured by YES bioassay and the EEQs calculated from chemical concentrations. These detected estrogenic compounds, such as 4-nonylphenol and estrone, were responsible for the estrogenic activities in the effluents. The genotoxic effects expressed as benzo[a]pyrene equivalent concentrations (BaP EQs) varied between below detection and 88.2 μg/L, with a mean of 8.76 μg/L in all effluents. The target polycyclic aromatic hydrocarbons were minor contributors to the genotoxicity in the effluents, and some nontarget compounds in the effluents were responsible for the measured genotoxicity. In terms of estrogenic activities and genotoxicity, discharge of these effluents could pose high risks to aquatic organisms in the receiving environments.     

Duplication 2p and monosomy 8p in mosaicism: clinical, molecular cytogenetic and molecular markers of a unique case

We report on a female patient, with a de novo mosaicism for a structural rearrangement producing trisomy 2p21-->pter and monosomy 8p21-pter. GTG bands and fluorescence in situ hybridization (FISH) in lymphocytes identified: mos 46,XX,der(8)(8qter-->8p21::p21::2p21-->pter),9qh +[52]/ 46,XX,9qh+[82]. Fibroblasts showed the same cell lines in 15 and 12 cells respectively. DNA profiling with fourteen autosomal STR markers, did not reveal a chimerism status in our patient. She did not present the classical phenotype described for trisomy 2p and for monosomy 8p probably due to approximately 60% of the patient's cells being normal. The abnormality probably arose in a very early stage of development during the first post-fertilization divisions with a non-sister chromatid exchange event between chromosomes 2 and 8 producing three cellular clones: a normal clone, one with trisomy 2p and monosomy 8p and a third with monosomy 2p and trisomy 8p. Only the first two cell lines were found in both lymphocytes and fibroblasts of hypopigmented skin; the third may have been lost or limited to other tissues.     

FISHing for answers: the use of molecular cytogenetic techniques in adolescent medicine practice

Chromosomal abnormalities are common causes of a variety of diseases, cancers, and malformation syndromes. Identification of chromosomal aberrations is important for counseling families about prognosis and reproductive risks with future pregnancies. However, limited resolution leads to the inability to detect small deletions, small insertions or duplications, and complex chromosomal rearrangements. Fluorescence-based assays, which have become possible because of sophisticated cloning technologies and improved sensitivity of antibody conjugates, enable the detection of subtle chromosomal changes beyond the resolution of classic cytogenetics. Such techniques have greatly expanded the diagnostic armamentarium available in the investigation of adolescents with mental retardation, malformations and many other disorders.     

Sickle cell anemia: a potential nutritional approach for a molecular disease

A certain population of red blood cells in patients with sickle cell anemia has an elevated density and possesses an abnormal membrane. These "dense cells" have a tendency to adhere to neutrophils, platelets, and vascular endothelial cells, and, thus, they could trigger vasoocclusion and the subsequent painful crisis from which these patients suffer. We developed a laboratory method of preparing such dense cells and found that nutritional antioxidant supplements, hydroxyl radical scavengers, and iron-binding agents could inhibit the formation of dense cells in vitro. The concentrations at which effective nutritional supplements could inhibit dense cell formation by 50% were 4.0 mg/mL for aged garlic extract, 0.38 mg/mL for black tea extract, 0.13 mg/mL for green tea extract, 0.07 mg/mL for Pycnogenol, 930 microM for alpha-lipoic acid, 270 microM for vitamin E, 45 microM for coenzyme Q(10), and 32 microM for beta-carotene. Both an ex vivo study and a pilot clinical trial demonstrated that a cocktail consisting of daily doses of 6 g of aged garlic extract, 4-6 g of vitamin C, and 800 to 1200 IU of vitamin E may indeed be beneficial to the patients.     

Original molecular cytogenetic approach to determining spontaneous chromosomal mutations in the interphase cells to evaluate the mutagenic activity of environmental factors

Standard cytogenetic methods are presently used to analyze chromosome anomalies in human somatic cells in order to evaluate the mutagenic activity of environmental factors. Their application is associated with the fact that uncultured cells cannot be analyzed. In this connection there is an urgent need to devise and introduce studies of the number and structure of interphase chromosomes. The paper describes the molecular cytogenetic approach to determining spontaneous chromosomal mutations in the interphase cells to evaluate the mutagenic activity of environmental factors, which is based on the original methods of interphase fluorescence in situ hybridization (FISH) and multicolor chromosome staining and on quantitative FISH and immune FISH. The proposed set of the methods has been tested examining 400000, 200000, and 1600000 embryonic, extraembryonic, and postnatal tissue cells, respectively. The analysis has yielded data on sporadic chromosomal mutations and shown that the proposed complex of the methods can be successfully used to evaluate the mutagenic activity of environmental factors leading to sporadic chromosomal mutations.     

Cytogenetic alterations induced by SPL (spent potliners) in meristematic cells of plant bioassays

Spent potliners (SPL) is solid waste generated by the aluminum industry during the manufacture of aluminum metal. Information on the cytotoxicity effect of SPL is necessary to facilitate understanding of their action on organisms and to subsidize environmentally correct solutions. Thus, the aim of the present investigation is to compare the effect of SPL on meristematic cells of Allium cepa and Zea mays and also to discuss the mechanisms of SPL cytotoxicity involved. A strong inhibition on root growth in higher SPL concentrations has been observed in both A. cepa and Z. mays. For cytogenetic analysis, the results showed a reduction of mitotic index and increase of different abnormalities as the SPL concentration increased. We observed bridges, chromosome fragments, stickiness, multipolar anaphase, later segregation and cell death. In general, it was possible to observe an increase of different abnormalities as the SPL concentration increased. It is obvious from the results of the present investigation that SPL is cytotoxic on meristematic cells of plant tests (A. cepa and Z. mays).     

Population cytogenetic and molecular evidence for existence of a new species in Anopheles fluviatilis complex (Diptera: Culicidae)

Anopheles fluviatilis James, an important malaria vector in the Oriental region has been established as a complex of at least three cryptic species which vary in their biological characteristics and malaria transmission potential. The sibling species S, T and U of Fluviatilis Complex can be identified by examination of species-specific fixed inversions in the polytene chromosomes and can also be differentiated by an allele-specific PCR assay based on differences in the D3 region of 28S ribosomal DNA (rDNA) of these species. Here we report a new An. fluviatilis population from villages under Laksar Community Health Centre, District Haridwar (Uttarakhand state), India which differs from the three sibling species of Fluviatilis Complex by two fixed paracentric inversions, s¹ and S in polytene chromosome arms 2 and 3 respectively. Longitudinal study carried out in study villages showed that the new cytotype was sympatric with species T and U in all the collections and no inversion heterozygotes were observed between them. Thus presence of two fixed paracentric inversions in polytene chromosomes with total absence of inversion heterozygotes demonstrates reproductive isolation which unequivocally establishes this cytological variant as a new species, provisionally designated as species V in the Fluviatilis Complex. Analysis of DNA sequences of D3 domain of 28S rDNA and ITS 2 region has also shown that species V is distinctly different from species S, T and U. With the discovery of new species in the Fluviatilis Complex, in-depth studies are required to know its distribution pattern and biological characteristics and to ascertain its role in malaria transmission.     

A plant life-cycle bioassay for contaminated soil,with comparison to other bioassays: Mercury and zinc

Bioassays using rapid-cycling plants allow measurement of multiple endpoints and assessment of impacts on both growth and reproduction. Selections of Brassica rapa develop rapidly in a broad range of soils and are very consistent in production of flower and seed. Their sensitivity to variation in growth conditions was investigated to define the variables that most affect performance. Yield differences between soils were substantial, indicating the need for careful selection and use of control treatments. The sensitivity to contaminants was investigated with applications of mercury (Hg) and zinc (Zn) to three soils. In a sand soil, bloom initiation was slowed by <10 mg Hg kg^–1 soil and <50 mg Zn kg^–1 soil. In contrast, lettuce emergence and earthworm survival were less sensitive to these metals in this soil. Survival of Daphnia magna and the Microtox^® assay in soil extracts were more sensitive to Hg than bloom initiation, but less sensitive to Zn. A similar relationship among the bioassays was observed for two finer-textured soils, although for these, effects were usually apparent only at soil metal concentrations >200 mg kg^–1. Enzyme assays were included for comparison, but were not sensitive to Hg contamination. Rapid-cycling B. rapa selections are suitable for routine bioassays, and are representative of several widely distributed and utilized species.     

Measuring precision in bioassays: Rethinking assay validation

The m:n:θ_b procedure is often used for validating an assay for precision, where m levels of an analyte are measured with n replicates at each level, and if all m estimates of coefficient of variation (CV) are less than θ_b, then the assay is declared validated for precision. The statistical properties of the procedure are unknown so there is no clear statistical statement of precision upon passing. Further, it is unclear how to modify the procedure for relative potency assays in which the constant standard deviation (SD) model fits much better than the traditional constant CV model. We use simple normal error models to show that under constant CV across the m levels, the probability of passing when the CV is θ_b is about 10% to 20% for some recommended implementations; however, for extreme heterogeniety of CV when the largest CV is θ_b, the passing probability can be greater than 50%. We derive 100q% upper confidence limits on the CV under constant CV models and derive analogous limits for the SD under a constant SD model. Additionally, for a post‐validation assay output of y, we derive 68.27% confidence intervals on either the mean or log geometric mean of the assay output using either y±s (for the constant SD model) or (for the constant CV model), where s and r_G are constants that do not depend on y. We demonstrate the methods on a growth inhibition assay used to measure biologic activity of antibodies against the malaria parasite.     

Assessing the genotoxicity of imidacloprid and RH-5849 in human peripheral blood lymphocytes in vitro with comet assay and cytogenetic tests

A combined approach employing comet assay and micronucleus (MN) and sister chromatid exchanges (SCE) tests was utilized to assess the genotoxicity of two pesticides, imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitro-imidazolidin-2-ylideneamine] and RH-5849 [2'-benzoyl-1'-tert-butylbenzoylhydrazine], on human peripheral blood lymphocytes in vitro. No significant difference in the frequencies of MN and SCE from the negative groups (P>0.05) was observed at low dose levels (i.e., 0.05 mg/L for imidacloprid and 5mg/L for RH-5849). As the concentrations of imidacloprid and RH-5849 were increased to 0.1 and 25 mg/L, respectively, significant effects to the frequencies of MN and SCE (P<0.05) were achieved relative to those of the negative controls. MN and SCE frequencies increased similarly in a dose-related manner with both pesticides. With the comet assay, however, the distribution of DNA damage grades in all the pesticide-treated groups was significantly different from those in the control (P<0.01). DNA damage scores increased with the exposure levels of both pesticides, and linear dose-effect relationships were observed for both imidacloprid (r2=0.98) and RH-5849 (r2=0.92). The cytogenetic techniques and comet assay revealed potential adverse effects of both imidacloprid and RH-5849 in human peripheral blood lymphocytes in vitro. Combination of the comet assay and cytogenetic tests appears commendable to assess the potential risks of human exposure to the pesticides.     

使用体内多终点遗传毒性检测体系评估2-甲基呋喃的遗传毒性

目的使用体内遗传毒性综合评价体系(Pig-a基因突变试验、流式微核试验和彗星试验)检测2-甲基呋喃的体内遗传毒性。方法 30只SPF级雄性SD大鼠分为6组,染毒组连续3 d经口灌胃染毒2-甲基呋喃25、50、100和150 mg/kg,溶剂对照为植物油,阳性对照为80 mg/kg N-乙基-N-亚硝基脲。于试验第3 d给药后3 h进行外周血彗星试验;试验第0(灌胃前1日)、14和28 d进行Pig-a基因突变试验;试验第0、4 d进行外周血微核试验。结果彗星试验中150 mg/kg组彗尾DNA百分含量显著升高; Pig-a基因突变试验结果显示,第14、28 d所有染毒组成熟红细胞和网织红细胞突变率均未见显著升高;流式微核试验所有染毒组网织红细胞微核率未见显著升高。结论该实验条件下,2-甲基呋喃急性暴露具有诱变性的可能性较低。