Journal of Toxicology and Environmental Health

ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19

Effect of carbon tetrachloride on hamster tracheal epithelial cells M. Ahmadizadeh , R. Echt , W. W. Heusner , L. M. Ross & R. A. Roth To cite this article: M. Ahmadizadeh , R. Echt , W. W. Heusner , L. M. Ross & R. A. Roth (1990) Effect of carbon tetrachloride on hamster tracheal epithelial cells, Journal of Toxicology and Environmental Health, 30:4, 273-285, DOI: 10.1080/15287399009531429 To link to this article: http://dx.doi.org/10.1080/15287399009531429

Published online: 20 Oct 2009.

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Date: 16 June 2016, At: 17:59

EFFECT OF CARBON TETRACHLORIDE O N HAMSTER TRACHEAL EPITHELIAL CELLS M. Ahmadizadeh, R. Echt

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Department of Anatomy Michigan State University, East Lansing, Michigan W. W. Heusner School of Health Education, Counseling Psychology and Human Performance, Michigan State University, East Lansing, Michigan L. M. Ross, R. A. Roth

Departments of Anatomy and Pharmacology and Toxicology, and Center for Environmental Toxicology, Michigan State University, East Lansing, Michigan

This study was designed to assess effects of carbon tetrachloride (CCl4) in hamster tracheal epithelium. Adult, male, Syrian golden hamsters were treated with 2.5 ml/kg CCl4 ip, and controls received only the vehicle (peanut oil). Animals were sacrificed after 1, 4, 12, and 24 h. Tissue samples from upper and lower tracheal levels were fixed and embedded in glycol methacrylate for light microscopy. Some tracheal rings were also fixed in formaldehyde/glutaraldehyde cacodylate buffer for transmission electron microscopy. For histopathologic evaluation of the tracheal epithelial cells, each tracheal level was cut transversely at 3 µm and stained with toluidine blue. CCl4 produced injury to ciliated and nonciliated cells in all portions of hamster trachea, although the severity of CCl4-induced injury differed in various levels and regions. The number of damaged cells increased markedly after 1 h in the lower trachea, but not until after 4 h in the upper trachea. By 24 h, the number of injured cells had decreased so that no significant difference from control was evident. The ultrastructural alterations in epithelial cells were obvious as early as 1 h after CCl4 administration. Intracellular organelles, including smooth and rough endoplasmic reticulum, mitochondria, and Colgi apparatuses, were damaged by this chemical. Since CCl4-induced cell injury is dependent on metabolism by intracellular NADPH-dependent cytochrome P450 monooxygenases, these results suggest that hamster tracheal epithelial cells have the potential to activate CCl4 metabolically.

This study was supported in part by NIH grants ES02581 and HL32244. The technical assistance of Dr. Ester Roege and Ms. Inge Taubitz in electron microscopy is gratefully appreciated. We thank Dr. Ernest Moore for using his facilities and Dr. Karen Klomparens for consultation on electron microscopy. We also thank Ms. Diane Hummel for her help in preparing the manuscript. Dr. R. Echt, the second author on this paper, is deceased. He contributed greatly to the conduct of the study throughout all phases prior to his untimely death. Requests for reprints should be sent to Dr. Robert A. Roth, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Ml 48824. 273 Journal of Toxicology and Environmental Health, 30:273-285, 1990 Copyright © 1990 by Hemisphere Publishing Corporation

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INTRODUCTION Cytochrome P-450 monooxygenases (MFOs) occur in smooth endoplasmic reticulum (SER) and provide a major pathway for metabolism of xenobiotic agents (Bend et al., 1972). Generally, the MFO system produces metabolites that are less toxic than the parent compounds. However, in some cases, MFO metabolism leads to the formation of metabolites more toxic than the parent compound. For example, carbon tetrachloride (CCI4) is metabolized by MFO components to highly cytotoxic free radical(s) (Recknagel and Glende, 1973). Cellular localization of cytochrome P-450-dependent monooxygenases in the respiratory system has generated considerable interest because many environmental chemicals gain access to the body through the respiratory system. In this respect, detoxification or activation of a chemical by the pulmonary MFO system may in part determine its site of action. Clara cells have been suggested as a primary site of MFO enzyme activity in the lung (Boyd, 1977; Serabjit-Singh et al., 1980). The presence of Clara-like cells has been identified in the trachéal epithelium of several species, including hamster, mouse, and rabbit (Hansell and Moretti, 1969; Pack et al., 1980; Al-Ugaily et al., 1980; Plopper et al., 1983). CCI4 produced injury to Clara cells of intrapulmonary airways in mice and rats (Boyd et al., 1980). It has been suggested that these cells are susceptible to CCI4 injury because of their capacity to metabolize this chemical to one or more toxic intermediates. Like intrapulmonary Clara cells, trachéal Clara cells contain abundant amount of smooth endoplasmic reticulum, and it seems likely therefore that a xenobiotic agent such as CCI4 might produce injury in trachéal epithelium. In testing this hypothesis, we examined the effect of CCI4 on various regions and levels of trachéal airway epithelium, since specific cell types differ in their distribution among portions of the trachea (Babero et al., 1973; Bivin et al., 1979; Jeffrey and Reid, 1975). MATERIALS AND METHODS Adult, male, Syrian golden hamsters (100-140 g) were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, Ind.) and housed in groups of 3 in clear polypropylene cages in a light cycle (12 h light and 12 h dark) and temperature-controlled room. The animals were allowed food (Wayne Lab Blox, Chicago, III.) and tap water ad libitum. Carbon tetrachloride (CCI4) was obtained from Sigma Chemical Company (St. Louis, Mo.), dissolved in peanut oil, and administered ip at a dose of 2.5 ml/kg body weight (i.e., 26 mmol/kg). This dose was chosen on the basis of its ability to injure bronchiolar Clara cells in rats and mice (Boyd et al., 1980). Control animals received the vehicle only. At 1, 4,12, or 24 h after treat-

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ment, the animals were killed with 100-300 mg/kg of sodium pentobarbital (¡p). Three hamsters were used for each group.

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Light Microscopy

Trachéal tissues were fixed in 4% paraformaldehyde in phosphate buffer (pH 7.2). Each specimen was divided into upper and lower levels and embedded in glycol methacrylate (GMA, Polyscience, Inc.). Tracheas were sectioned transversely at 3 /¿m from proximal to distal ends with glass knives on a jB-4 microtome. Three histological sections, each at least 15 /¿m apart, were taken from each tissue block and stained with 1% toluidine blue. The luminal epithelium was subdivided into dorsal, right and left lateral, and ventral regions. The total number of injured cells per millimeter of epithelium in each region was counted at 400 x magnification and compared statistically. Only those cells with distinct nuclei were counted. The criteria for cell injury included nuclear dilation, loss of staining capacity, and obvious cellular swelling. Data were expressed as mean ± standard error. The results were analyzed by analysis of variance, completely randomized design, and treatment differences were identified by the method of Newman-Keuls (Steel and Torrie, 1960); p < .05 was used as the criterion for significance. Transmission Electron Microscopy

One or two rings of trachea from each animal were cut into pieces approximately 1 mm2, and 1-2 blocks from each region (dorsal, lateral, or ventral) were prepared for electron microscopy. Tissues were fixed in formaldehyde/glutaraldehyde cacodylate buffer (Karnovsky, 1965). The tissues were washed in 0.2 M cacodylate buffer, postfixed in 2% osmium tetroxide, dehydrated with ethanol, and embedded in Epon-araldite (Mollenhauer, 1964). Sections 1 /¿m thick were cut and stained with toluidine blue for examination by light microscopy. The sections (approximately 90 nm) were cut from selected areas. The sections were stained with uranyl acetate and lead citrate and examined with a Philips 201 transmission electron microscope. RESULTS Administration of peanut oil vehicle alone did not produce detectable injury to hamster trachéal epithelial cells (Fig. 1). However, cell injury was observed in the various levels and regions of the trachea following CCI4 treatment. Light microscopy revealed that both ciliated and nonciliated trachéal epithelial cells were swollen and had loss of staining capacity, and had dilated nuclei. CCI4 produced injury in trachéal epithelial cells as early as 1 h following CCI4 treatment (Fig. 2). However, the

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FIGURE 1. Light micrograph of hamster trachea treated with vehicle only (control). The ciliated cells (open arrow) and nonciliated cells (solid arrow) are intact. x400.

FIGURE 2. Light micrograph of trachéal epithelial cells of hamster treated 1 h previously with CCI4. Loss of staining capacity, dilatation of the nucleus and cellular swelling in ciliated cells (open arrow) and nonciliated cells (solid arrow), are evident. X400.

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TABLE 1. Numbers of Damaged Cellsa in Various Levels and Regions of Trachéal Epithelium of Hamsters Treated with CCI 4 b Time after CCI4 administration Control Dorsal region UT 0 ± 0

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LT

0 ± 0

Lateral region UT 0±0 LT

0 + 0

Ventral region UT 0± 0 LT

0± 0

1h

4h

12 h

24 h

2.3 ± 1.3e-' (1.6) 18.1 ± 8.3 (13.0)

18.5 ± 4.7 c ' d (13.2) 1.3 ± 0.4 (0.9)

13.3 ± 1.8c'd (9.5) 4.3 ± 0.9 (3.1)

2.2 ± 0.6e-' (1.6) 0.7 ± 0.1 d (0.5)

1.3 ± 0.5e'f (0.9) 11.7 ± 4.4 (8.3)

15. ± 5.2c-d (10.7) 3.2 ± 1.1' (2.3)

12.8 ± 0.9c'd (9.1) 18.5 ± 5.3c-e (13.2)

3.2 ± 0.6e&,

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Effect of carbon tetrachloride on hamster tracheal epithelial cells.

This study was designed to assess effects of carbon tetrachloride (CCl4) in hamster tracheal epithelium. Adult, male, Syrian golden hamsters were trea...
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