Lead is perhaps the longest used and best recognized toxic environmental chemical and it is still being used recklessly. Lead (Pb) has been found to be capable of eliciting a positive response in an extraordinarily wide range of biological and biochemical tests; among them tests for enzyme inhibition, fidelity of DNA synthesis, mutation, chromosomal aberrations, cancer and birth defects. Since inhalation is one of the most important routes of environmental Pb exposure, in the present study a lead inhalation model in mice was implemented in order to detect the induction of genotoxic damage as single-strand breaks and alkali-labile sites in several mouse organs (nasal epithelial cells, lung, whole blood, liver, kidney, bone marrow, brain and testes), assessed by single cell gel electrophoresis (SCGE) or Comet assay. We found differences among the organs studied after a single and subsequent inhalations: in the organs analyzed we observed a positive induction of DNA damage after a single inhalation only in the liver and the lung. In subsequent inhalations the response was positive in all organs except the testicle, however, DNA damage induction over time was different for each organ. A correlation between length of exposure, DNA damage and metal tissue concentration was observed for lung, liver and kidney. Differences in DNA damage occurred in organs when lead acetate was administered acutely or sub-chronically. These results show that lead acetate inhalation induces systemic DNA damage but that some organs are special targets of this metal, such as lung and liver, depending in part on length of exposure, suggesting alternative organ processes to handle lead intoxication.

/pdf/genotoxicity-induced-in-cd-1-mice-by-inhaled-lead-1bkx7dcrwq.pdf

Genotoxicity induced in CD-1 mice by inhaled lead: differential organ response.

Vanadium (V) has increased in the air as a component of suspended particles originated from fuel combustion. In this report, a model of inhaled V in mice was implemented to identify the effect that V has in the corpus striatum and substantia nigra, structures with high concentrations of dopamine and scarce antioxidants burden. Mice inhaled 0.02 M V2O5 1 h twice a week and were sacrificed at points from 1 to 8 weeks after inhalation, perfused, and processed for Golgi method and for tyroxine hidroxylase (TH) inmunocytochemistry. Cytological analysis consisted in counting the number of dendritic spines in 20 medium-size spiny neurons and the number of TH immunoreactive neurons in the substatia nigra pars compacta. Dendritic spine density decreased drastically after V exposure; the same was observed with the TH-positive neurons, which decreased in a time-dependent mode. No previous morphological studies about V and nervous system have been reported. The decrease in spine density and in TH-positive neurons might have functional repercussions that should be studied because the trend of this element in the atmosphere is to increase.

Nigrostriatal modifications after vanadium inhalation: an immunocytochemical and cytological approach.

Spatial memory may be severely impaired as a consequence of ageing and neurodegenerative diseases, conditions that include neuronal damage. Vanadium (V) is a metalloid widely distributed in the environment and exerts severe toxic effects on a wide variety of biological systems. Reports about V inhalation toxicity on the CNS are limited, thus the purpose of this study is to determine the effects of Vanadium pentoxide (V(2)O(5)) inhalation (0.02M) on the memory and its correlation with the cytology of the hippocampus CA1. Forty eight CD-1 male mice were trained in spatial memory tasks and inhaled 1h twice a week; after each inhalation animals were evaluated and sacrificed from 1 to 4 weeks, perfused and processed for Golgi method and for ultrastructure evaluation. The cytological analysis consisted in counting the number of dendritic spines of 20 pyramidal neurons of hippocampus CA1, as well as ultrastructural characteristics. Results show that V inhalation produces a time dependent loss of dendritic spines, necrotic-like cell death, and notorious alterations of the hippocampus CA1 neuropile, which correlate with spatial memory impairment. Our data suggest that V induces important cellular and functional alterations, fact that deserves special attention since the concentration's trend of this element in the atmosphere is increasing.

Hippocampal cell alterations induced by the inhalation of vanadium pentoxide (V2O5) promote memory deterioration

Ultrastructural modifications in the mitochondrion of mouse Sertoli cells after inhalation of lead, cadmium or lead-cadmium mixture.

The purpose of this study was to examine concentrations of selected heavy metals in the liver and kidney of brown hares (Lepus europaeus). In addition, correlations between heavy metals and biochemical parameters in blood plasma were determined. The average concentrations of heavy metals (mmol/L) +/- SD were as follows: liver: Pb 0.221 +/- 0.189, Cd 0.160 +/- 0.140, Hg 0.021 +/- 0.030, kidney: Pb 0.115 +/- 0.125, Cd 1.570 +/- 1.103, Hg 0.030 +/- 0.053. The average concentrations of biochemical parameters in the blood plasma were as follows: Ca 3.16 mmol/L, P 2.19 mmol/L, Mg 1.40 mmol/L, Na 148.71 mmol/L, K 8.12 mmol/L, glucose 6.56 mmol/L, total proteins 56.49 g/L, urea 5.00 mmol/L, total lipids 1.40 g/L, bilirubin 3.97 micro mol/L, cholesterol 1.53 mmol/L, aspartate aminotransferase (AST) 6.06 micro kat/L and alanine aminotransferase (ALT) 1.94 micro kat/L. Average levels of hormones (ng/mL) were as follows: testosterone 2.94, androstendiol 0.13, estradiol 501.59, progesterone 6.63, oxytocin 328.60. Tissue analysis showed an accumulation of lead, cadmium and mercury in the liver and kidney of brown hares. There were no significant correlations between levels of heavy metals in liver, kidney, and biochemical parameters.

Environmental levels of cadmium, lead and mercury in brown hares and their relation to blood metabolic parameters

Fortoul, T. I . , R. C. Salgado, S. G. Moncada, I. G. Sanchez, I. E. Lopez, G. Espejel, N. L. Calderon, L.Saldivar: Ultrastructural Findings in the Murine Nonciliated Bronchiolar Cells (NCBC) after Subacute Inhalation of Lead Acetate. Acta Vet. Brno 1999, 68: 51-55. Air pollution is an important health problem in some countries. For Mexico City, repeatedly high levels of some metals including lead (Pb) have been reported. Since there is no relevant information, we used an inhalation model to identify the possible lung cell damage after exposure of animals to Pb. We used thirty CD-1 male mice that were inhaling (for 1 hour) aerosolized lead acetate 0.1M, three times per week during two weeks. Mice were sacrificed by cervical dislocation on days 1, 3, 5, 7, 10 and 15 after the last Pb exposure. Their lungs were fixed by intratracheal instillation of glutaraldehyde. Lung tissue for morphological observation and metal concentration was sampled. In the exposed mice, on days 3 and 5 changes in the nonciliated bronchiolar cells appeared. Whorl-like structures were present in the cell apex. These structures compressed other organelles. On day 5 after the final inhalation, the structures increased in size, and by day 10 they disappeared. After the last inhalation, the metal concentration in the lung tissue continuously decreased until day 7 when no more metal was detected. It was evident that the contact of the NCBC with Pb produces changes in the morphology of these cells. The metal concentrations in the lung decreased when the exposure ended. This finding supports the assumption that Pb is not accumulated in lung tissue. As a consequence, the cellular modifications decreased and began their way to morphological recovery. Nonciliated bronchiolar cell, Clara cell, Pb, inhalation toxicology, bronchiole Air pollution is a major problem worldwide, particularly in areas such as Mexico City that is considered the most polluted city in the world. In some industrial areas of this city, air concentrations of lead (Pb) ranged from 7.5 to 14.5μg/m3 (three months average) as reported by Alber t and Badi l lo (1992). A report from our laboratory showed increased concentrations of Pb and other metals in human autopsied lungs when comparing a thirty-year period of the records of people who lived in Mexico City during the 1950’s to those of the ones who lived in the same city but in the 1980’s (For toul 1996). Lead is a component of the 0.1 to 1 μm size particles present in the air that enters the lungs by inhalation. Thirty to 50 % of Pb concentration in this organ is absorbed into blood and from here it is distributed into the whole organism. In situations when the exposure to polluted air is high, the dose by this route could be a great risk resulting in lead intoxication (Tsa lev 1985). Only few reports on Pb toxicity for lung tissue are available, and they mostly focus only on modifications in the function of the alveolar macrophages ACTA VET. BRNO 1999, 68: 51–55 Address for correspondence: Teresa I. Fortoul, M. D. Departamento de Biologia Celulary Tisular Edif. A 3er piso Facultad de Medicina UNAM CP 04510, Mexico City, Mexico Phone: +525 623-2360 Fax: +525 623-2182 or 623-2399 E-mail: fortoul@servidor.unam.mx (Zel ikof f 1993). Changes in the function of the immune system with decreased antibacterial resistance have also been mentioned (Ehr l ich 1980). Almost all written information available is about the effects of this metal on the hematopoietic system, on the kidney or on the nervous system. Lack of information exists as to the effects of inhalation of this metal directly in the lungs and changes in the morphology of cells involved in the detoxification of xenobiotics. The nonciliated bronchiolar cells (NCBC) are located in the ultimate conducting tubes of the lung; they are rich in cytochrome P-450 monooxigenases and they are the target of a number of xenobiotics including furans, aromatic and halogenated aliphatic hydrocarbons, ozone and others (Kanekal 1990; Serabj i t -Singh 1979; Boyd 1977). This cell type could also be a natural target for inhaled lead. We therefore conducted experimental Pb aerosol exposures using a mouse model to examine the morphologic changes in the nonciliated bronchiolar cells. Materials and Methods Lead acetate in deionized water was tested at concentrations of 0.001M, 0.01M and 0.1M. Light microscopy of the lungs revealed no changes with the first two concentrations, and only slight changes were observed with 0.1M. With these results it was decided to start the experiments using 0.1M concentration, and a schedule at which the animals would survive. Mice were selected as a model animal for their evident NCBC (Plopper 1991), and to make sure that the suspected target cells would be produced in such quantity, that the modifications would be easily detected. For one hour three times a week for two weeks, thirty CD-1 male mice weighing 35 ± 5 g inhaled a solution 0.1M of lead acetate in deionized water. The mice were placed in a plastic chamber volume of 72.7 l to which an ultrasonic nebulizer (Ultra Neb 99, DeVilbis) was attached. The nebulizer was designed to produce droplets in a 0.5 to 5 μm range. A trap for the vapor was located on the opposite side with a solution of sodium bicarbonate to precipitate the remaining Pb. Eighteen mice of the same weight were used as controls, inhaling solely the vehicle with the same schedule. As a second control for acetate, sodium acetate 0.1M was also inhaled by a group of twelve mice. After each exposure, mice were placed in plastic cages, and they had free access to water and food (Purina Chow) in 12 hour day and light cycles. After the final inhalation, mice were sacrificed by cervical dislocation on day 1, 3, 5, 7, 10 and 15 according to the local standard legislation for observing animal welfare. Immediately after cervical dislocation, the trachea was exposed and the lungs were fixed by intratracheal instillation with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.4 (470 mmol) at total lung capacity (TLC). After lung expansion, the trachea was tied up and the cardiopulmonary block was removed from the chest cavity. The left lower lobe was ligated and processed for transmission electron microscopy (TEM) following the usual technique in order to identify the terminal bronchiole (Sabat ini 1964). Determinat ion of Pb in the t issue We quantified the metal using a modified technique (Fortoul 1996). Results were defined as μg/g per dry lung tissue (EPA 1994; Keith 1983). Determinat ion of Pb concentrat ion in the inhaled air A filter was positioned at the outlet of the nebulizer, used in the entire experiment, for sixty minutes at a flow rate of 10 l/min. The 400 ml solution used during the entire experiment was nebulized. The filter was removed from the outlet after ten minutes of exposure and then weighed. The treatment of the filter was the same as that of the tissue samples. Using the fluxes dynamic equation C=M/V in which C was the concentration of Pb in the filtered air expressed as μg/m3; M corresponded to the total lead mass on the filter, expressed as μg. The volume V in m3 was V = Q*t in which Q was the flow rate in l/ min and t the time exposure expressed in minutes. Lead concentration in the filter was 7180 μg . The Volume (10 l/ min * 60 min.) = 0.06 m3 . Concentration of Pb in the inhaled air was 11966.6 μg/m3.

/pdf/ultrastructural-findings-in-the-murine-nonciliated-4altwo05pr.pdf

S. G. Moncada

Papers

Ultrastructural Findings in the Murine Nonciliated Bronchiolar Cells (NCBC) after Subacute Inhalation of Lead Acetate