Evaluation of the genotoxic potential of cypermethrin, chlorpyrifos and their subsequent mixture, on cultured bovine lymphocytes.

Cypermethrin (CYP) and chlorpyrifos (CPF) are insecticides/parasiticides used in the production of fruits, vegetables and beef cattle. These substances or their metabolites are frequently reported as residues in food, whose consumption in a diet implies a genotoxic risk. The potential for chronic toxicity of CYP and CPF is unclear, and only a few genotoxicological evaluations based on their mixture have been performed. The aim of this study was to evaluate the genotoxic potential of CYP, CPF and CYP + CPF in five concentrations, from 5.9 to 175 µg/mL, on bovine lymphocytes. By means of the cytokinesis-block micronucleus cytome assay, a decrease in the cell proliferation index was observed (r = -0.89 p = 0.04); and also an increase in the frequencies of binucleated cells (BN) with micronuclei (BNMn) (r = 0.93, p = 0.02) and BN with nuclear buds (BNBud) (r = 0.778 p = 0.04), depending on the concentrations of CPF. An increase in BNMn frequencies was observed as a function of CYP concentrations (r = 0.89, p = 0.04) and also of the CYP + CPF mix (r = 0.99, p = 0.008). CYP caused greater genotoxic damage (BNMn) than CPF and the mixture on bovine lymphocytes. Cells with simultaneous presentation of micronuclei and nuclear buds were detected, as well as cells with irregular nuclei, something never previously reported, whose origin and significance should be investigated. The genotoxic effect of chlorpyrifos, cypermethrin and their mixture on bovine lymphocytes was observed. We recognized the value of the use of primary bovine cultures, animal species adjacent to man in the food chain, for genotoxicity studies.

Micronucleus assay as a biomarker of genotoxicity in the occupational exposure to agrochemicals in rural workers.

This paper aims to evaluate the genotoxic effect of agrochemicals in rural workers occupationally exposed by the micronucleus assay in peripheral blood lymphocytes and to promote the development of health and environmental preventive and protective practices. A total of 30 blood samples from 20 individuals occupationally exposed to different agrochemicals and 10 unexposed persons, who formed the reference group, were analyzed. We found statistically significant differences (p < 0.0005, Student's t Test) in the frequency of micronuclei between the two groups (7.20 ± 1.55 and 15.15 ± 5.10 CBMN for reference and exposed groups respectively). The analysis of age showed a positive correlation (Pearson Correlation Test) with the frequency of micronuclei in exposed population (p < 0.05; r(2) = 0.47), in contrast with smoking habits and years of exposure. Micronucleus assay allows an early detection of populations at higher risk of having genetic damage, allowing us to implement strategies of intervention for the purpose of contributing to reduce that risk.

Genotoxicity of AMPA, the environmental metabolite of glyphosate, assessed by the Comet assay and cytogenetic tests.

Formulations containing glyphosate are the most widely used herbicides in the world. AMPA is the major environmental breakdown product of glyphosate. The purpose of this study is to evaluate the in vitro genotoxicity of AMPA using the Comet assay in Hep-2 cells after 4h of incubation and the chromosome aberration (CA) test in human lymphocytes after 48h of exposition. Potential in vivo genotoxicity was evaluated through the micronucleus test in mice. In the Comet assay, the level of DNA damage in exposed cells at 2.5-7.5mM showed a significant increase compared with the control group. In human lymphocytes we found statistically significant clastogenic effect AMPA at 1.8mM compared with the control group. In vivo, the micronucleus test rendered significant statistical increases at 200-400mg/kg. AMPA was genotoxic in the three performed tests. Very scarce data are available about AMPA potential genotoxicity.

Antioxidant capacity of vitamin C in mouse liver and kidney tissues

In the present study, the antioxidant capacity of vitamin C was examined in the liver and the kidney tissues of mice with or without ciprofloxacin (CFX) treatment. The antioxidant capacity of the vitamin was evaluated in terms of lipid hydroperoxides (LOOH) and thiobarbituric acid reactive substances (TBARs). The experimental design was 15 days of water (control and CFX groups) or vitamin C (vitamin C and vitamin C plus CFX groups) in drinking water. One dose of CFX was injected, 15 minutes before sacrifice, in the corresponding mice. The initial nmol of lipid hydroperoxides/g of tissue were 137 +/- 11 in the kidney and 145 +/- 15 in the liver, and the nmol of TBARs were 13 +/- 0.7 and 12 +/- 0.6, respectively.Pre-treatment with vitamin C reduced the levels of LOOH in the liver to 45 +/- 11 (p < 0.01) and vitamin C with CFX injection to 54 +/- 9 (p < 0.01). Vitamin C treatment also reduced the LOOH levels in the kidney roughly duplicated by CFX. Through the TBARs method we have not observed these effects. Quantification of LOOH is more sensitive than that of TBARs for estimating lipid peroxidation. CFX is used especially for urinary infections and can produce oxidative stress in the kidney. Pre-treatment with vitamin C may ameliorate this stress and also may improve the oxidative balance in the liver.

Antioxidant capacity of vitamin C in mouse liver and kidney tissues

In the present study, the antioxidant capacity of vitamin C was examined in the liver and the kidney tissues of mice with or without ciprofloxacin (CFX) treatment. The antioxidant capacity of the vitamin was evaluated in terms of lipid hydroperoxides (LOOH) and thiobarbituric acid reactive substances (TBARs). The experimental design was 15 days of water (control and CFX groups) or vitamin C (vitamin C and vitamin C plus CFX groups) in drinking water. One dose of CFX was injected, 15 minutes before sacrifice, in the corresponding mice. The initial nmol of lipid hydroperoxides/g of tissue were 137 +/- 11 in the kidney and 145 +/- 15 in the liver, and the nmol of TBARs were 13 +/- 0.7 and 12 +/- 0.6, respectively.Pre-treatment with vitamin C reduced the levels of LOOH in the liver to 45 +/- 11 (p < 0.01) and vitamin C with CFX injection to 54 +/- 9 (p < 0.01). Vitamin C treatment also reduced the LOOH levels in the kidney roughly duplicated by CFX. Through the TBARs method we have not observed these effects. Quantification of LOOH is more sensitive than that of TBARs for estimating lipid peroxidation. CFX is used especially for urinary infections and can produce oxidative stress in the kidney. Pre-treatment with vitamin C may ameliorate this stress and also may improve the oxidative balance in the liver.(AU)

Increase of tissue lipid hydroperoxides as determination of oxidative stress.

Increased levels of lipid hydroperoxides (LOOH) are frequently associated with the oxidative mechanisms involved in physiological states as ageing and with serious pathological conditions. In the present work the physiological and the CCl4-induced lipid hydroperoxides levels in mice liver and kidney were determined. The analysis of LOOH tissue levels was performed through the oxidation of 1-napthyldiphenylphosphine (NDPP) into its oxide (ONDPP) and further quantification by high pressure liquid chromatography at 292 nm UV detection. The physiological level of lipid hydroperoxides levels was higher in the kidney (245 +/- 8 nmol LOOH/g of tissue) than in liver (164 +/- 5 nmol of LOOH/g tissue). After a single administration of CCl4 (0.25 ml/g) tissue LOOH reached a maximum level after 15 min (416 +/- 21 nmol/g kidney and 303 +/- 6 nmol/g liver) and then slowly decreased. LOOH levels in liver afforded an early indicator (15 min) of oxidative damage. LOOH levels in kidney remained significatively increased up to 60 min post administration. The described HPLC assay is a useful, simple and sensitive method to detect cellular oxidative stress and damage.

Increase of tissue lipid hydroperoxides as determination of oxidative stress

Increased levels of lipid hydroperoxides (LOOH) are frequently associated with the oxidative mechanisms involved in physiological states as ageing and with serious pathological conditions. In the present work the physiological and the CCl4-induced lipid hydroperoxides levels in mice liver and kidney were determined. The analysis of LOOH tissue levels was performed through the oxidation of 1-napthyldiphenylphosphine (NDPP) into its oxide (ONDPP) and further quantification by high pressure liquid chromatography at 292 nm UV detection. The physiological level of lipid hydroperoxides levels was higher in the kidney (245 +/- 8 nmol LOOH/g of tissue) than in liver (164 +/- 5 nmol of LOOH/g tissue). After a single administration of CCl4 (0.25 ml/g) tissue LOOH reached a maximum level after 15 min (416 +/- 21 nmol/g kidney and 303 +/- 6 nmol/g liver) and then slowly decreased. LOOH levels in liver afforded an early indicator (15 min) of oxidative damage. LOOH levels in kidney remained significatively increased up to 60 min post administration. The described HPLC assay is a useful, simple and sensitive method to detect cellular oxidative stress and damage.

Increase of tissue lipid hydroperoxides as determination of oxidative stress

Increased levels of lipid hydroperoxides (LOOH) are frequently associated with the oxidative mechanisms involved in physiological states as ageing and with serious pathological conditions. In the present work the physiological and the CCl4-induced lipid hydroperoxides levels in mice liver and kidney were determined. The analysis of LOOH tissue levels was performed through the oxidation of 1-napthyldiphenylphosphine (NDPP) into its oxide (ONDPP) and further quantification by high pressure liquid chromatography at 292 nm UV detection. The physiological level of lipid hydroperoxides levels was higher in the kidney (245 +/- 8 nmol LOOH/g of tissue) than in liver (164 +/- 5 nmol of LOOH/g tissue). After a single administration of CCl4 (0.25 ml/g) tissue LOOH reached a maximum level after 15 min (416 +/- 21 nmol/g kidney and 303 +/- 6 nmol/g liver) and then slowly decreased. LOOH levels in liver afforded an early indicator (15 min) of oxidative damage. LOOH levels in kidney remained significatively increased up to 60 min post administration. The described HPLC assay is a useful, simple and sensitive method to detect cellular oxidative stress and damage.(AU)

Increase of tissue lipid hydroperoxides as determination of oxidative stress.

Increased levels of lipid hydroperoxides (LOOH) are frequently associated with the oxidative mechanisms involved in physiological states as ageing and with serious pathological conditions. In the present work the physiological and the CCl4-induced lipid hydroperoxides levels in mice liver and kidney were determined. The analysis of LOOH tissue levels was performed through the oxidation of 1-napthyldiphenylphosphine (NDPP) into its oxide (ONDPP) and further quantification by high pressure liquid chromatography at 292 nm UV detection. The physiological level of lipid hydroperoxides levels was higher in the kidney (245 +/- 8 nmol LOOH/g of tissue) than in liver (164 +/- 5 nmol of LOOH/g tissue). After a single administration of CCl4 (0.25 ml/g) tissue LOOH reached a maximum level after 15 min (416 +/- 21 nmol/g kidney and 303 +/- 6 nmol/g liver) and then slowly decreased. LOOH levels in liver afforded an early indicator (15 min) of oxidative damage. LOOH levels in kidney remained significatively increased up to 60 min post administration. The described HPLC assay is a useful, simple and sensitive method to detect cellular oxidative stress and damage.

Intravenous pharmacokinetics of ciprofloxacin in goats.

The pharmacokinetics of ciprofloxacin in goats was studied following intravenous administration. A single dose of 10 mg/kg was administered as an intravenous bolus, and serum samples were collected at predetermined intervals over a 24 h period. Ciprofloxacin levels were measured using high pressure liquid chromatography and the resulting concentration versus time curve was analyzed using a non linear regression analysis. The data were best represented by a two-compartment pharmacokinetic model with a mean elimination half-life of 2.72+/-1.04 h. Mean pharmacokinetic parameters obtained were area under the curve (AUC: 10.320+/-5.137 microg/ml per h), mean residence time (MRT: 3.334+/-1.428 h), apparent volume of distribution (Vdss: 3.373+/-0.893 l/kg) and total body drug clearance (tCl 19.596+/-9.059 ml/min per kg). Ciprofloxacin in goats showed the general pharmacokinetic characteristics of a typical fluoroquinolone antibacterial agent and we recommend a dose of 10 mg/kg to be administered intravenously at 12 h intervals to goats.

Comparative pharmacokinetics of enrofloxacin and ciprofloxacin in chickens.

The pharmacokinetics of enrofloxacin (EFX) and ciprofloxacin (CFX) was investigated in broiler chickens. Each antimicrobial was administered intravenously at a dose of 5 mg/kg body weight. Blood was taken in different preset times: prior and at 0.03, 0.06, 0.13, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h following drug administration. The concentrations of EFX and CFX in plasma were determined by high pressure liquid chromatography (HPLC). Plasma concentrations vs. time were analysed by a compartmental independent pharmacokinetic model that provided the most important kinetic parameters. Statistically significant differences between the two antimicrobials were found for most of the pharmacokinetic parameters: Area under the curve (AUC), area under first moment curve (AUMC), mean residence time (MRT), total body cleareance (ClB), volume of distribution beta (Vd beta) and volume of distribution at the steady state (Vd(ss)). Both antimicrobials were widely distributed in chickens throughout the body with a mean Vd(ss) of 1.98+/-0.18 L/kg for EFX, and 4.04+/-0.69 L/kg for CFX. The ClB for CFX was five times higher than that obtained for EFX. AUC, MRT and the diminished half time for EFX were two-four times higher than those obtained for CFX. These results indicate that CFX remains in the body for less time than the other quinolone. This characteristic of CFX suggests the advantage of a shorter withdrawal time for food producing animals treated with this antimicrobial.

Chromosomal aberrations in human lymphocytes exposed in vitro to enrofloxacin and ciprofloxacin.

Chromosomal aberrations were evaluated in cultures of human peripheral lymphocytes from eight healthy donors, exposed to the antimicrobial enrofloxacin (EFX) or to its major metabolite ciprofloxacin (CFX). In both treatments cultures revealed an increase in the chromosomal aberration level, detected as chromatid and chromosome breaks and gaps. Control cultures analysis revealed 3.6 +/- 0.6 chromosomal aberrations per 100 cells while treated cultures exhibited 8.3 +/- 0.8 and 9.6 +/- 1.2 aberrations at 5 and 50 microg/ml of EFX respectively. In CFX treated cultures it was found 5.6 +/- 1.3 and 7.7 +/- 3.5 aberrations/100 cells at 5 and 25 microg/ml antimicrobial concentration. These results suggested a genotoxic effect of EFX and CFX in the system used (P < 0.001). A reduction in the mitotic index and fuzzy metaphases were observed at 50 microg/ml of CFX, indicating a cytotoxic effect produced by this antimicrobial.

Cytogenetic characterization of an extra structurally abnormal chromosome associated with severe mental retardation: inv dup (15) (q13).

We have studied an extra structually abnormal chromosome (ESAC) in a 13 years old boy with profound mental, psychomotor and speech retardation, behavioral problems, seizures and abnormal electroencephalogram. The examination of the bisatellited ESAC with chromosome banding demonstrated that the karyotype was: 47, XY, +inv dup (15) (pter-->q13::q13-->pter). The cytogenetic characterization of the inv dup (15) is reported with special emphasis on the usefulness of DA/DAPI staining when G-banding is sequentially performed to discard possible heteromorphisms in DA/DAPI positive chromosomes, and the importance of Ag-NOR heteromorphisms to ascertain the maternal origin of the inv dup (15). A U-type exchange between two non-sister chromatids is proposed as its mechanism of formation. The clinical features of the case were consistent with those previously reported in similar cases.

HPLC residues of enrofloxacin and ciprofloxacin in eggs of laying hens.

Eggs of 12 laying hens with 5 mg/kg/day oral administration of 5% enrofloxacin (EFX) or ciprofloxacin (CFX) solution during 5 days contained residues from 0.02 to 1.98 microg/g (EFX) or 0.14 to 0.28 microg/g (CFX). At identical dosage regime High Performance Liquid Chromatograhy (HPLC) residues of EFX were 6-fold greater than CFX ones. Maximun concentrations were detected at the second day after the administration withdrawal. The limits of detection were 0.019 microg/g for EFX and 0.156 microg/g for CFX. The recovery was 36-50% for CFX and 49-85% for EFX. The withdrawal treatment periods in hens are six days for EFX and five days for CFX in order to avoid violative levels of egg residues.

Chromosomal damage in workers occupationally exposed to chronic low level ionizing radiation.

Chromosomal aberrations were evaluated in cultures of peripheral lymphocytes from subjects working in diagnostic X-ray and nuclear medicine areas, exposed to electromagnetic ionizing radiation and particulate ionizing emissions, respectively. A 4-fold increase in the level of chromosomal aberrations was found between the exposed and control groups without qualitative or quantitative cytogenetic differences between X-rays and nuclear medicine-exposed workers. Results are discussed in view of the early damage detection from chronic exposures particularly related to biological controls, hygienic improvements and overwork in a developing country.

Non-random distribution of spontaneous chromosome aberrations in two Bloom Syndrome patients.

The distribution of breakpoints involved in spontaneous chromosome aberrations (CA) was analyzed in lymphocytes from a family with Bloom's Syndrome (BS) and 9 healthy individuals. Standard and G-banded metaphases from each individual were analyzed to allow the identification of the breakpoints involved in spontaneously occurring chromosome aberrations. A total of 85 breakpoints in BS patients, 17 in their parents and 35 in controls, could be exactly localized to specific chromosome bands. Breakpoint distribution was statistically analyzed considering the formula proposed by Brøgger (1977), showing a non-random pattern in BS patients. Thirteen bands non-randomly involved in spontaneous CA (p < 0.005) were recognized in BS, located at 1p36, 1q21, 1q32, 2q33, 3p24, 3p14, 3q27, 5q31, 6p21, 7q22, 9q13, 11q13, and 17q23. Only 1 band (1q21) was significantly implicated in both parents (p < 0.005), while controls showed a random distribution. BS non-random bands were correlated with the chromosomal location of fragile sites, oncogenes, and breakpoints involved in cancer rearrangements. A significant correlation with the location of fragile sites and cancer-breakpoints (p < 0.005), particularly with acute myeloid leukemia and malignant lymphomas rearrangements was found. These findings demonstrate that constitutional chromosome instability in BS might involve specific points, such as fragile sites and cancer breakpoints, suggesting an association with the increased incidence of cancer.

Spontaneous chromosome aberrations in Fanconi's anemia patients are located at fragile sites and acute myeloid leukemia breakpoints.

Spontaneous chromosome aberrations (CA) were analyzed in 3 Fanconi's anemia (FA) patients, 8 family members, and 9 healthy individuals. Peripheral blood lymphocytes obtained from each individual were cultured and cytogenetic analysis was performed on standard and sequential G-banded metaphases. The numbers of abnormal cells and breaks were found to be higher in AF patients compared to the other groups (p < 0.0001). Breakpoint distribution was statistically analyzed considering the formula proposed by Brøgger (1977), showing a non-random pattern among FA patients but not among controls or relatives (p < 0.001). Five chromosomal bands located at 1p36, 1p22, 1q21, 3p14, and 3q21 were non-randomly involved in spontaneous CA in FA patients. These bands were correlated with the chromosomal location of fragile sites, oncogenes, and breakpoints involved in cancer-rearrangements. A significant correlation with the location of fragile sites (p < 0.03) and breakpoints involved in cancer-rearrangements (p < 0.001), particularly with AML chromosome anomalies (p < 0.03) was found, suggesting a possible relationship with the high predisposition to cancer observed in this disease.

Increased expression of 5q31 fragile site in a Bloom syndrome family.

In this work, we report spontaneous chromosomal breakpoints and fragile site expression induced by 5-fluorodeoxyuridine (FdUrd) and FdUrd plus caffeine in a family with Bloom's syndrome (BS) and 2 healthy donors. Standard and G-banded metaphases from each individual and each treatment were analyzed. Among the 59 common fragile sites (c-fra) identified in this work, only the frequency of 5q31 was significantly increased in the BS family with respect to healthy donors (P less than 0.005). A remarkable coincidence between the breakpoints involved in spontaneous chromosome aberrations and induced c-fra was found in BS homozygote patients. The importance of the interaction between fragile sites and chromosome rearrangements in cancer is discussed.

Lack of sensitivity of sister-chromatid exchange for lymphocyte chromosomal damage detection caused by antichagasic treatment.

No studies exist on sister-chromatid exchange (SCE) formation in chagasic patients therapeutically exposed to nifurtimox (NFX) or benznidazol (BZ). In the present study SCE was analyzed in cultures of peripheral lymphocytes of patients aged 11 months to 11 years treated with NFX 12-15 mg/kg/d for 60 days or with BZ 5 mg/kg/d for 30 days. Chagasic patients before treatment constituted a control group. A mean of 30 metaphases were examined for each individual. All treated patients compared with untreated controls did not show a significant increase in SCE frequency. Compared with the percentage of chromosomal aberrations in these patients and others belonging to the same epidemic protocol, SCE seems to be less sensitive in the detection of lymphocyte chromosomal damage caused by NFX or BZ.