Stability studies in biological fluids during post-analysis custody. Opiate compounds derived from heroin consumption.

In Forensic Toxicology, the evidences have to be maintained under custody for, at least, one year. Depending on the conditions and duration of storage, drug concentrations might have changed considerably since the first analysis. The aim of this study is to evaluate in vitro stability of opiate compounds, derived from heroin consumption, 6-acetylmorphine (6-MAM), morphine (MOR) and codeine (COD), in blood and urine, during post-analysis custody. Parameters evaluated were: time of custody, temperature, addition of preservative (blood) and pH (urine). Blood and urine samples were spiked with the three analytes to give a final concentration of 1000 ng/mL. The prepared samples were divided into 2 groups and stored at two temperatures (4 °C and -20 °C). Each one of these groups was subsequently divided in other two groups: with and without preservative (1%NaF) for blood, and pH 4 and 8 in the case of urine. 6-MAM, MOR and COD were analyzed by GCMS after SPE and derivatization with BSTFA. Analyses were performed in triplicate every two weeks for a year. In blood samples 6-MAM is the only compound that degrades. The best storage conditions were at -20 °C with NaF, with 6-MAM recoveries, after one year of custody, of 47.1 ± 1.5%; while in the other conditions 6-MAM disappeared after 215 days (at 4 °C with NaF), 45 days (at -20 °C without NaF) and 15 days (at 4 °C without preservative). COD does not degrade, with recoveries higher than 90%, in all of the conditions. They ranged from 89.7 ± 3.6% in samples maintained at -20 °C without NaF to 95.9 ± 2.0% in those maintained at 4 °C with NaF. MOR recoveries were lower than those of COD. They ranged from 66.9 ± 3.6%, in frozen samples added with NaF, to 78.6 ± 0.5% in refrigerated samples without preservative. In urine samples the three compounds were stable in all the studied conditions, with the exception of 6-MAM in samples at pH 8 and stored at 4 °C. In these conditions, 6-MAM disappeared after 135 days of custody; while recoveries in the other conditions ranged from 93.7 ± 6.4%, at 4 °C and pH 4, to 85.1 ± 2.0% at -20 °C and pH 8. MOR and COD recoveries were similar in the four conditions. In the case of MOR, they ranged from 82.1 ± 1.2% at 4 °C and pH 4 to 89.5 ± 6.0% at -20 °C and pH 8. As far as COD is concerned, recoveries ranged from 111.6 ± 5.8% at 4 °C and pH 8 to 102.6 ± 1.2% at 4 °C and pH 4. In conclusion, the study showed that the most labile opiate compound is 6-MAM. Its stability mainly depends on urine pH or the addition of preservative, in blood samples. The best storage conditions for samples from heroin consumers are in the freezer, at -20 °C. In addition, blood samples must be added with 1%NaF and urine samples must be buffered at pH 4.

[Study of oxidative stress in advanced kidney disease]. / Estudio del estrés oxidativo en enfermedad renal avanzada.

INTRODUCTION: Introduction Patients with Chronic renal Disease (CRD) often have cardiovascular disease that is the main cause of morbidity and mortality. Oxidative stress and a subclinical inflammation are crucial factors in its development. The aim of this study was to asses the oxidation of the main molecular lines in patients with advanced renal disease without dialysis and to determinate the best biomarker to asses this stress. PATIENTS AND METHODS: We performed an observational study to measure the most important oxidative biomarkers in 32 patients with stage 4 CRD (MDRD = 22.1 +/- 1.08 ml/min) compared with the values obtained in a control group. In peripheral lymphocytes we measured, the lipid peroxidation by Malondialdehyde (MDA) and F2 Isoprostanes in plasma; protein oxidation by glutathione oxidized/reduced ratio (GSSG/GSH) in peripheral lymphocytes and protein carbonyls in plasma and the oxidative damage in genetic material by modified nucleotide base 8-deoxiguanosina oxo -(8-oxo-dG), after isolating nuclear and mitochondrial DNA. We also studied the antioxidant defenses with superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GSR) and catalase (CAT) in peripheral lymphocytes. We studied the correlation between oxidative stress and the renal function and oxidative stress and co-morbidity factors. RESULTS: All biomarkers showed important differences in comparison with the control subjects. 821.89 +/- 300.47 ng/ml vs. 270 (95.66) * ng/ml (p < 0.000), MDA 0.11 (0.11) * vs. 0.7 +/- 0.31 nmol/mg prot (p <0.000). GSSG / GSH: 6.89 +/- 1.91 vs. 1.39 +/- 0.75 (p <0.000), protein carbonyls: 7.41 +/- 0.84 vs. 3.63 (1.12) *. Nuclear 8-oxo-dG 7.88 (2.32) vs. 2.96 (1.78) * mitochondrial 8-oxo-dG: 15.73 +/- 2.28 vs. 13.85 +/- 1.44 (p <0.05). The Antioxidant enzymes also showed differences. Nuclear 8-oxo-dG demonstrated an important relationship with the rest of biomarkers, homocysteine (r = 0.305, p <0.05), lipoprotein (a) (r = 0.375, p <0.01), mitochondrial 8-oxo-dG (r = 0.411, p <0.05), GSSH/GSH (r = 0.595, p <0.001) and protein carbonyls (r = 0.489, p <0.05). There was an inverse correlation with total protein (r = -0.247, p <0.01), GSH (r = -0.648, p <0.000), GSR (r = -0.563, p <0.001) and SOD (r = -0.497, p <0.000). We did not find any correlation between these parameters and renal function. The presence of diabetes or the treatment with statins did not showed significant differences. * Median (Interquartile range). CONCLUSION: There is an important oxidative stress in patients with advanced renal disease, probably established during early stages of disease. Of the studied parameters, the nuclear 8-oxo-dG is the best marker for oxidative stress in CRD.

Aquatic toxicity assessment of the additive 6-methylcoumarine using four experimental systems.

The toxicity assessment of chemicals is one of the main issues in the current policies in order to protect the health of the environment and human beings. Food and cosmetic additives have been extensively studied in relation to their toxicity to humans, but data about their ecotoxicological effects are scarce. The aim of this study was to evaluate the toxic effects of the additive 6-methylcoumarine in the aquatic milieu using a test battery comprising experimental model systems from different trophic levels. The inhibition of bioluminiscence was studied in the bacteria Vibrio fischeri (decomposer), the inhibition of growth was evaluated in the alga Chlorella vulgaris (producer) and immobilization was studied in the cladoceran Daphnia magna (first consumer). Finally, several end points were evaluated in the RTG-2 salmonid fish cell line, including neutral red uptake, protein content, methylthiazol tetrazolium salt metabolization, glucose-6-phosphate dehydrogenase activity, lactate dehydrogenase activity and leakage, and morphology. The sensitivity of the test systems employed was as follows: V. fischeri > D. magna > C. vulgaris > RTG-2 cell line. The results show that 6-methylcoumarine is not expected to produce acute toxic effects on the aquatic biota. However, chronic and synergistic effects with other chemicals cannot be excluded and should be further investigated.

Acute and subacute toxic effects produced by microcystin-YR on the fish cell lines RTG-2 and PLHC-1.

Approximately 80 microcystins (MCs) variants have been isolated in surface water worldwide. The toxicity of the most frequently MCs are encountered, MC-LR and MC-RR, has been extensively studied in humans and animals. However, studies dealing with MC-YR toxicity are still scarce. In this work, the toxic effects of MC-YR were investigated in the fish cell line PLHC-1, derived from a hepatocellular carcinoma of the topminnow Poeciliopsis lucida, and RTG-2 fibroblast-like cells derived from the gonads of rainbow trout Oncorhynchus mykiss. After 48 h, morphological and biochemical changes (total protein content, neutral red uptake and methylthiazol tetrazolium salt metabolization) were determined. The most sensitive endpoint for both cell lines was the reduction of total protein content, with EC(50) values of 35 microM for PLHC-1 cells and 67 microM for the RTG-2 cell line. Lysosomal function and methylthiazol tetrazolium salt metabolization were stimulated at low concentrations, while they decreased at high doses. Increase of piknotic cells, rounding effects, reduction in cell number and cell size, hydropic degeneration, and death mainly by necrosis but also by apoptosis were observed in the morphological study. Furthermore, PLHC-1 cells are more sensitive than RTG-2 cells to MC-YR exposure. These results were similar to those obtained when both cell lines were exposed for 24h to a Microcystis aeruginosa isolated strain extract containing MC-LR.

Toxic effects produced by microcystins from a natural cyanobacterial bloom and a Microcystis aeruginosa isolated strain on the fish cell lines RTG-2 and PLHC-1.

Toxic cyanobacterial blooms are a worldwide problem, causing serious water pollution and public health hazard to humans and livestock. The intact cells as well as the toxins released after cellular lysis can be responsible for toxic effects in both animals and humans and are actually associated with fish kills. Two fish cell lines-PLHC-1 derived from a hepatocellular carcinoma of the topminnow Poeciliopsis lucida and RTG-2 fibroblast-like cells derived from the gonads of rainbow trout Oncorhynchus mykiss were exposed to several concentrations of extracts from a natural cyanobacterial bloom and a Microcystis aeruginosa-isolated strain. After 24 hours, morphologic and biochemical changes (total protein content, lactate dehydrogenase leakage, neutral red uptake, methathiazole tetrazolium salt metabolization, lysosomal function, and succinate dehydrogenase [SDH] activity) were investigated. The most sensitive end point for both cyanobacterial extracts in PLHC-1 cells was SDH activity, with similar EC(50) values (6 microM for the cyanobacterial bloom and 7 microM for the isolated strain). RTG-2 cells were less susceptible according to SDH activity, with their most sensitive end point lysosomal function with an EC(50) of 4 microM for the M. aeruginosa-isolated strain and 72 microM for the cyanobacterial bloom. The lysosomal function was stimulated at low concentrations, although SDH activity increased at high doses, indicating lysosomal and energetic alterations. Increased secretion vesicles, rounding effects, decreased cell numbers and size, hydropic degeneration, esteatosis, and apoptosis were observed in the morphologic study. Similar sensitivity to the M. aeruginosa-isolated strain was observed in both cell lines, whereas the cyanobacterial bloom was more toxic to the PLHC-1 cell line.

The use of the fish cell lines RTG-2 and PLHC-1 to compare the toxic effects produced by microcystins LR and RR.

Cyanobacterial toxins, especially microcystins (MC), are found in eutrophied waters through the world. Acute poisonings of animals and humans has been reported following MC exposure. In the present study, two fish cell lines, PLHC-1 and RTG-2, were evaluated after exposure to the cyanobacterial toxins MC-LR and MC-RR. The effects of different concentrations of the toxins were investigated in both cell lines at morphological and biochemical levels (total protein content, lactate dehydrogenase leakage, lysosomal activity and succinate dehydrogenase activity). The results obtained showed a decrease in protein content and no relevant increase in cell disruption, except for MC-LR in PLHC-1 cells. Morphological changes produced by microcystins were cellular swelling, blebbling, rounding, reduction in the cell number and increase in the number and size of lysosomal bodies. In addition, steatosis was produced in hepatoma PLHC-1 cells, particularly with MC-RR. Furthermore, the fish PLHC-1 cell line was more sensitive than RTG-2 cells to the cyanobacterial toxins compared, being the stimulation of the lysosomal function and the induction of steatosis the most specific changes detected.

Ecotoxicological evaluation of diethanolamine using a battery of microbiotests.

In order to investigate the potential ecotoxicity of diethanolamine (DEA), a battery of model systems was developed. DEA is widely used as a chemical intermediate and as a surface-active agent in cosmetic formulations, pharmaceuticals and agricultural products. DEA was studied using ecotoxicological model systems, representing four trophic levels, with several bioindicators evaluated at different exposure time periods. The battery included bioluminescence inhibition of the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris and immobilization of the cladoceran Daphnia magna. Cell morphology, total protein content, neutral red uptake, MTS metabolization, lysosomal function, succinate dehydrogenase activity, G6PDH activity, metallothionein levels and EROD activity were studied in the hepatoma fish cell line PLHC-1, derived from Poeciliopsis lucida. The systems most sensitive to DEA were both D. magna and V. fischeri, followed by C. vulgaris and the fish cell line PLHC-1. The most prominent morphological effect observed in PLHC-1 cultures exposed to DEA was the induction of a marked steatosis, followed by death at high concentrations, in some cases by apoptosis. The main biochemical modification was a nearly three-fold increase in metallothionein levels, followed by the stimulations of lysosomal function and succinate dehydrogenase and G6PDH activities. Judging by the EC(50) values in the assay systems, DEA is not expected to produce acute toxic effects in the aquatic biota. However, chronic and synergistic effects with other chemicals cannot be excluded.

Ecotoxicological evaluation of carbamazepine using six different model systems with eighteen endpoints.

The occurrence of pharmaceutically active compounds in the aquatic environment has been recognized as one of the emerging issues in environmental chemistry. However, the ecotoxicological effects of pharmaceuticals have still not been researched adequately. Carbamazepine, an anticonvulsant commonly present in surface and groundwater, was studied, using six ecotoxicological model systems with eighteen endpoints evaluated at different exposure time periods. The battery included the immobilization of Daphnia magna, bioluminescence inhibition in the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris, and micronuclei induction and root growth inhibition in the plant Allium cepa. Cell morphology, neutral red uptake, total protein content, MTS metabolization, lactate dehydrogenase leakage and activity and glucose-6-phosphate dehydrogenase activity were studied in the salmonid fish cell line RTG-2. The total protein content, LDH activity, neutral red uptake and MTT metabolization in Vero monkey kidney cells were also investigated. The most sensitive system to carbamazepine was the Vero cell line, followed by Chlorella vulgaris, Vibrio fischeri, Daphnia magna, Allium cepa, and RTG-2 cells. EC50 values from 19 microM in Vero cells at 72 h to more than 1200 microM in other systems, were obtained. Comparing the concentrations in water and the toxicity quantified in our assay systems, carbamazepine is not expected to produce acute toxic effects in the aquatic biota under these circumstances, but chronic and synergistic effects with other chemicals cannot be excluded.

Tribromophenol induces the differentiation of SH-SY5Y human neuroblastoma cells in vitro.

Tribromophenol is a pesticide with fungicide activity, presently used as a replacement of pentachlorophenol as a wood preservative, and as a flame retardant in electronic and electrotechnical devices. Retinoic acid differentiated and non-differentiated SH-SY5Y human neuroblastoma cell cultures were exposed to a range of concentrations of tribromophenol for 24, 48 and 72 h and the effects evaluated at morphological, basal cytotoxicity and biochemical levels. Neuroblastoma cell number, evaluated by quantification of total protein content, was increasingly inhibited in accordance with the concentration of tribromophenol and the exposure time period. According to the mean effective concentrations, differentiated cultures were nearly three times more sensitive than naive cells. Lysosomal function evaluated by the neutral red uptake was stimulated, particularly in non-differentiated cells. MTS metabolization was stimulated by all the treatments, with more potency at 24 h for differentiated cells. Acetylcholinesterase activity increased with the time of exposure in non-differentiated cells, while in differentiated cells the activity was doubled at 24 h. Morphological alterations were evident from 12.5 microM, showing hydropic degeneration and reduction in cell number, and from that concentration, piknosis and apoptotic bodies were observed. In conclusion, the main effects detected for tribromophenol were the induction of neuroblastoma cell differentiation, as expressed by the inhibition of cell growth and the increase in acetylcholinesterase activity with a critical cell concentration of 0.1 microM. Apoptosis was observed at high concentrations. The induction of cell differentiation and the special sensitivity of differentiated cells can explain some mechanisms involved in the embryotoxic and foetotoxic potential of tribromophenol.

A test battery for the ecotoxicological evaluation of pentachlorophenol.

Experimental bioassays are currently used in ecotoxicology and environmental toxicology to provide information for risk assessment evaluation of new chemicals and to investigate their effects and mechanisms of action; in addition, ecotoxicological models are used for the detection, control and monitoring of the presence of pollutants in the environment. As a single bioassay will never provide a full picture of the quality of the environment, a representative, cost-effective and quantitative test battery should be developed. The effects of pentachlorophenol were studied using a battery of ecotoxicological model systems, including immobilization of Daphnia magna, bioluminiscence inhibition in the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris, and micronuclei induction in the plant Allium cepa. The inhibition of cell proliferation and MTT reduction were investigated in Vero cells. Neutral red uptake, cell growth, MTT reduction, lactate dehydrogenase leakage and activity were studied in the salmonid fish cell line RTG-2, derived from the gonad of rainbow trout. Pentachlorophenol was very toxic for all biota and cells. The system most sensitive to pentachlorophenol, was micronuclei induction in A. cepa, followed by D. magna immobilization, bioluminescence inhibition in V. fischeri bacteria at 60 min and cell proliferation inhibition of RTG-2 cells at 72 h. Inhibition of cell proliferation and MTT reduction on Vero monkey cells showed intermediate sensitivity.

[Demographic aspects in indigenous communities of 3 regions of Colombia]. / Aspectos demográficos en comunidades indígenas de tres regiones de Colombia.

OBJECTIVE: To obtain demographic indicators for some indigenous communities in Colombia situated in three different regions of the country: the Caribbean, the Amazonian basin and the Andes. MATERIAL AND METHODS: Demographic variables gathered in a KAP (knowledge, attitude and practices) survey among the indigenous population in 1993 and 1994 were analyzed. The survey included 11,522 Indian. RESULTS: 45% of the population is under age 15; overall rate of fertility is 6.5 children per woman, and death rate in 1990 was 63.3 children per 1,000 live births. Life expectancy at birth was 57.8 years for women and 55.4 years for men. CONCLUSIONS: The indicators differ substantially from the national figures. Although the indigenous population seems to be undergoing a process of demographic transition, there are marked differences between regions, with significantly higher fertility and infant mortality rates for the Caribbean region.

Morphological, biochemical and molecular effects of cocaine on mouse neuroblastoma cells culture in vitro.

In order to compare the effects of cocaine at morphological, basal cytotoxicity, biochemical and molecular levels, cultured mouse neuroblastoma cells (Neuro-2a) were exposed to a range of concentrations of cocaine hydrochloride. Neuroblastoma cell proliferation, evaluated by quantification of total protein content, was very sensitive to cocaine, being increasingly inhibited from 12 to 72 hr of exposure (EC(50) = 3.1 mm at 24 hr). Cytoplasmic membrane permeability to lactate dehydrogenase was not particularly increased and lysosomal function was stimulated from 0.05 to 1.5 mm, and inhibited from 2.5 mm. A shift to anaerobiosis was detected as intracellular lactate dehydrogenase (LDH) activity was increased and mitochondrial succinate dehydrogenase (SDH) activity decreased. Hexosaminidase (HEX), a lysosomal enzyme involved in sphingolipid degradation, was stimulated only at 1 mm and neural acetylcholinesterase (AChE) activity was stimulated from 2.5 mm. Morphological examination of exposed cultures revealed that most cells became bipolar and multipolar neurons by extension of neurites, but also suffered cytoplasmic vacuolization, hydropic degeneration and nuclear pyknosis. Although cells developing apoptosis were observed, no DNA oligonucleosomal fragmentation was detected by agarose gel electrophoresis of DNA from cells exposed to cocaine. In conclusion, in addition to predominance of anaerobiosis, little disruption of membranes and severe morphologic injury, biochemical and morphological differentiation-like effects were the most prominent alterations produced by cocaine on mouse neuroblastoma cells.

[Mortality: vital statistics, regulation, and analysis]. / La mortalidad: estadisticas vitales, reglamentacion y analisis.

PIP: The author assesses the quality and coverage of death reporting in Colombia. In spite of deficiencies in vital statistics registration, the data indicate much about mortality trends in the country. Two major trends are noted: an increase in violent deaths among young adults, and a decline in mortality due to transmittable and non-transmittable diseases.^ieng

Karyometric marker features in fine needle aspirates of microinvasive follicular carcinoma of the thyroid.

Karyometric measurements were performed on fine needle aspirates of clearly identifiable tumor areas and adjacent normal-appearing areas in the surgical specimens from ten patients with microinvasive follicular carcinoma of the thyroid. Similar measurements were performed on aspirates from nine patients free of thyroid disease (controls). A total of 95 karyometric features were evaluated for each nucleus. As compared with the control nuclei, the normal-appearing nuclei showed a 6% increase in total nuclear optical density (OD) while the tumor nuclei showed a 14% increase. Analysis of variance indicated a significant difference between the normal-appearing nuclei and the control nuclei, with most of the difference due to the differences of tissue origin. Discriminant analysis selected nine features that produced a statistically highly significant separation of tumor nuclei from control nuclei. A similar analysis selected five features that produced a statistically highly significant discrimination of normal-appearing nuclei from control nuclei; the validity of those karyometric features as markers of malignancy in normal-appearing nuclei from tissues adjacent to microinvasive follicular carcinomas of the thyroid was demonstrated by analysis in further training and test sets of nuclei.

Mucinous metaplasia to neoplastic lesions in endometrial samples with cytohistologic correlation.

Seven cases of endometrial mucinous metaplasia and five of well-differentiated mucinous adenocarcinoma of the endometrium were studied. Cytologic specimens were obtained by Isaacs endometrial sampler, avoiding endocervical contamination. The histologic diagnosis between the intermediate type of mucinous metaplasia and the well-differentiated mucinous adenocarcinoma posed no problems. DNA analyses of the histologic samples showed a euploid pattern in benign and intermediate types of metaplasia, while well-differentiated mucinous adenocarcinoma showed a hyperdiploid pattern. The cytologic diagnosis of benign mucinous metaplasia should be suggested in the presence of abundant mucinous cells in endometrial samples in the absence of nuclear abnormalities.