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

TLDR: An inhalation model used to identify the possible lung cell damage after exposure of animals to Pb 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 live in the same city but in the 1980s.

Abstract: 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.