Effects of Propylene Oxide Exposure on Rat Nasal Respiratory Cell Proliferation

Melva N. Ríos-Blanco*, Shuji Yamaguchi{dagger}, Mukta Dhawan-Robl{ddagger}, Winfried Kessler{ddagger}, Robert Schoonhoven{dagger}, Johannes G. Filser{ddagger} and James A. Swenberg*,{dagger},1

* Curriculum in Toxicology and {dagger} Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and {ddagger} Institute of Toxicology, GSF National Research Center for Environment and Health, D-85764 Neuherberg, Germany

Received April 9, 2003; accepted June 25, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Long-term exposure of rodents to propylene oxide (PO) induced inflammation, respiratory cell hyperplasia, and nasal tumors at concentrations >= 300 ppm, suggesting a possible role for cytotoxicity and compensatory cell proliferation in PO carcinogenesis. In this study, the effects of PO exposure on histopathology and cell proliferation in nasal and hepatic tissues were studied in male F344 rats exposed by inhalation for 3 or 20 days (0, 5, 25, 50, 300, and 500 ppm). Histopathology revealed an increase in mucous cell hyperplasia in the anterior nasal passages after 20 days of exposure (>=300 ppm). This was associated with the formation of goblet cell nests. Cell proliferation was measured in the respiratory epithelium (NRE; mucociliary and transitional) lining the anterior nasal passages, the nasopharyngeal meatus (NPM), and the liver using BrdU administered with 3-day osmotic pumps. Significant increases in cell proliferation occurred (>3.6-fold) in the mucociliary epithelium lining the anterior nasal cavity at and above 300 ppm for both exposure periods. In the mucociliary epithelium, the 20-day labeling was commonly associated with nests of goblet cells. Significant increases in cell proliferation (>2.3-fold) were observed in the transitional epithelium at 500 ppm after 3 days of exposure and at 300 and 500 ppm after 20 days of exposure. Significant increases in cell proliferation in the NPM (>2.8-fold) were evident at 500 ppm PO after 3 days and at 300 and 500 ppm PO after 20 days of exposure. No exposure-related changes in cell proliferation were observed in the liver. These studies demonstrate a clear concordance between the site and exposure concentration for tumor induction and those causing significant increases in cell proliferation in the rat nose.

Key Words: propylene oxide; nose; rat; cytotoxicity; cell proliferation; inhalation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Propylene oxide (PO) is a high volume chemical intermediate used for the production of polyurethane foams, resins, and propylene glycol. Another major use is in the preparation of hydroxypropylcelluloses and sugars, surface-acting agents and isopropanolamine. Human exposure to PO can occur by inhalation in the workplace. An 8-h time-weighted average of 20 ppm was established for PO by the Occupational Safety and Health Administration in the U.S. At present, a notice for intended change of the current regulated limits to 2 ppm PO has been proposed. The German Technical Exposure Limit to PO is 2.5 ppm. PO is not known to occur naturally in the environment. Releases of PO into the atmosphere can occur during its production, transport, and use as a chemical intermediate.

PO is a direct DNA alkylating compound that induced mutations in bacterial and mammalian systems in vitro (reviewed by Ehrenberg and Hussain, 1981Go; Giri, 1992Go; Ríos-Blanco et al., 1997Go), but has not been shown to be mutagenic in vivo (Bootman et al., 1979Go; Hardin et al., 1983Go), with the exception of an increase in micronucleated erythrocytes of mice exposed ip to a high dose (300 mg/kg body weight) of the compound (Bootman et al., 1979Go) and an increase in sister chromatid exchanges in mouse bone marrow cells at ip doses of 300 and 450 mg/kg body weight (Farooqi et al., 1993Go). Monkeys exposed by inhalation to 300 ppm PO for 2 years had no increases in chromosome aberrations or sister chromatid exchanges (Lynch et al., 1984bGo).

Long-term inhalation exposure of rodents to PO led to a low incidence of nasal tumor formation in high exposure groups (>=300 ppm) only. F344 rats developed papillary adenomas (3–7% incidence) of the respiratory epithelium in the anterior nasal passages, while exposure to B6C3F1 mice led to the development of hemangiomas (8% incidence), hemangiosarcomas (7%), and adenocarcinomas (2%) within the nose (Lynch et al., 1984aGo; NTP, 1985Go; Renne et al., 1986Go). Histopathological examination of nasal tissues in the inhalation studies revealed that nasal respiratory cell hyperplasia and rhinitis accompanied tumor formation, and that these lesions were minimal or absent in rodents exposed to lower concentrations (100 and 200 ppm) and in controls. Inhalation exposure of Wistar rats to PO (0, 30, 100, and 300 ppm for 124 weeks) resulted in an increase in nonneoplastic lesions in the nasal cavity of rats in groups exposed to 100 or 300 ppm (Kuper et al., 1988Go). One squamous cell carcinoma of the nose was diagnosed in a 30 ppm male (1/61) and another in a 300 ppm male (1/63). Long-term intragastric administration of PO to rats (Dunkelberg, 1982Go) and sc injections in mice (Dunkelberg, 1979Go) led to the development of tumors in the forestomach of rats and in the subcutis of mice, suggesting that PO is a site of contact carcinogen.

Although differences in tumorigenic responses occurred between rodent species and strains, a common observation in all long-term inhalation studies was the significant increase in tissue inflammation and nasal cell hyperplasia at high concentrations of PO. According to these observations, an increase in cell replication secondary to cytotoxicity may play a promoting role in the induction of tumors after exposure to high concentrations of the compound and hence could be a biologically meaningful endpoint for PO risk assessments. Cell replication measurements in the rodent nasal epithelium, along with the examination of changes in other biologically relevant endpoints, such as detoxication and DNA damage, at exposures known to result in tumor formation, as well as those to which humans may be exposed, may provide critical insights for PO risk assessment. Such mechanistic data should improve the understanding and accuracy of risk estimates derived for humans from rodent bioassays.

Short-term studies (1–4 weeks) in F344 rats exposed to PO by the inhalation route (0, 10, 20, 50, 150, and 525 ppm) demonstrated an increase in cell hyperplasia in the nasal respiratory epithelium (NRE) in animals exposed to 525 ppm for 20 days (Eldridge et al., 1995Go). Examination of rats killed 1 or 4 weeks postexposure demonstrated that the increase in cell hyperplasia was reversible. A no-observed-adverse-effect level (NOAEL) of 50 ppm was established for nonneoplastic changes in nasal epithelium of rats after inhalation exposure to PO. In the aforementioned study, analysis of cell proliferation demonstrated a significant increase in cell replication in the NRE only in groups of rats exposed to 525 ppm for 1 or 4 weeks, thus the NOAEL for cell proliferation was established at 150 ppm PO.

The objectives of the present study were to investigate the relationship between (1) sites susceptible to neoplastic transformation in the rat bioassays and sites exhibiting significant increases in cell proliferation, (2) concentration responses for PO-induced nonneoplastic changes in the nose and the tumor incidence curve, and (3) the tumor incidence curve and the concentration response for cell proliferation in nasal tissue. We also examined differences in cell proliferation response between target and nontarget tissues for carcinogenesis after exposure of rats to PO for 3 or 20 days. Prior to conducting these studies, one of the authors (J.A.S.) visited the National Toxicology Program archives and reviewed the slides from the rat carcinogenicity bioassay (NTP, 1985Go) to determine the specific location of each of the nasal adenomas. The nasal adenomas were all located adjacent to the transitional epithelium lining the ventral lateral surface of the nasoturbinates.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
PO (99.99%) was provided by Lyondell Chemie Nederland (Rotterdam, Netherlands). Bromodeoxyuridine (BrdU) and Dulbecco’s phosphate buffered saline (pH 7.0) were from Sigma Co. (Deisenhofen, Germany).

Animals and propylene oxide exposures.
The study was conducted in compliance with good laboratory practice regulations as promulgated by the U.S. Environmental Protection Agency and the Organization for Economic Cooperation and Development. Nine-week old male F344 rats were purchased from Charles River Deutschland (Sulzfeld, Germany) and were marked with ear tags. The rats were assigned to one of six exposure groups per exposure period (n = 6/group) on the basis of their body weight. The group assignments were adjusted to result in mean group body weights that were not significantly different from one another. Treated and control animals were provided with food (Standard chow 1324—Altromin, Lage, Germany) and water ad libitum except during the inhalation exposure periods. Exposures were carried out at the GSF Institute of Toxicology, Neuherberg, Germany. The animals were exposed to nominal PO concentrations of 0, 5, 25, 50, 300, and 500 ppm, for 6 h/day, 5 days/week for 3 or 20 days. PO vapor was generated by a single vaporization system (Filser et al., 1993Go) using a separate glass chamber for each concentration. The actual measured mean chamber PO concentrations (± SD) were 0, 4.8 ± 0.4, 24.3 ± 1.7, 50.7 ± 2.6, 273 ± 21, and 500 ± 42 ppm for the three-day exposure, and 0, 5.1 ± 0.8, 24.4 ± 1.6, 52.9 ± 3.9, 291 ± 31, and 508 ± 54 ppm for the 20-day exposure. Exposures took place during the day (beginning around 0900 h) for 6 h/day, 5 days/week. Animals were observed daily for mortality and overt clinical signs of toxicity. Detailed descriptions of the exposure procedures, analytical techniques and inhalation chambers are found elsewhere (Filser et al., 1993Go; Ríos-Blanco et al., 1997Go).

Administration of BrdU.
Alzet osmotic pumps, Model 2ML (flow rate = 10 µl/h) were obtained from Alza Corp (Palo Alto, CA). Osmotic pumps containing a sterile solution of BrdU (20 mg/ml in Dulbecco’s phosphate buffered saline, pH 7.0) were surgically implanted sc over the thoraco-lumbar area of rats under isofluorane anesthesia (1.5–2%) using aseptic techniques 3 days prior to necropsy. Osmotic pumps were implanted in rats at the end of the day. The animals were observed until they were fully awake. Each rat was housed separately following insertion of the pumps. BrdU is a thymidine analog that it is incorporated into DNA during S phase of the cell cycle.

Tissue preparation.
Rats were euthanized under CO2 within 1 to 5 h following the last exposure to PO. Whole livers were removed and randomly selected longitudinal 3–4 mm midsections of the left, median, and right lobes were put in tissue cassettes (Fisher Scientific) and placed in a jar with 10% neutral phosphate-buffered formalin (NBF; Sigma Co., Germany). A section of duodenum (~5 cm) was also obtained from each animal and fixed in NBF. The head of each animal was removed, and the eyes, lower jaw, skin, and musculature were removed. The nasal airways were flushed with 10–15 ml NBF by means of a blunt 16 ga needle (attached to a 20 cc syringe) inserted approximately 5 mm into the posterior opening of the pharyngeal duct. The dissected head was then placed in a jar with fresh NBF along with the rest of the tissues collected. Tissues were fixed in NBF for 4.5 days. After fixation, tissues were transferred to jars filled with phosphate buffered saline (PBS; pH 7.4) and shipped to the University of North Carolina at Chapel Hill, where they were prepared for histopathology and immunohistochemistry. Dissected heads were individually wrapped in gauze and decalcified in a solution of formic acid with ion-exchange resin (Immunocal, Decal Corporation, Congers, NY) for 3 days. The dissected heads were rinsed in tap water for 15 min, immersed in a solution of Cal-Arrest (Decal Chem. Co., Congers, NY) for 20 min, rinsed in tap water overnight, and then transferred to a solution of 70% ethanol. After decalcification, the nasal passages were transversely sectioned at six specific anatomic locations (Fig. 1AGo). The locations selected are similar to those recommended by Young (1981)Go with the exception that one additional tissue block was evaluated in this study. The nasal tissue blocks were embedded in paraffin with the anterior face down. Liver slices were also embedded in paraffin. A piece of duodenum was included on each tissue block to confirm delivery of BrdU to all tissues. Tissue sections (5-µm thick) were cut from the anterior face of each nasal tissue block and from liver. Tissue sections were histochemically stained with hematoxylin and eosin following standard procedures and examined by light microscopy. Extra slides from each tissue section were also prepared for BrdU immunohistochemistry. Tissue slices were placed on 3-aminopropyltriethoxysilane-coated slides to ensure adhesion of the sections during immunostaining. Figures 1BGo and 1CGo demonstrate cross section diagrams of the tissue blocks selected for histopathology and BrdU analysis.



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FIG. 1. (A) Ventral surface of the skull of a F344 rat. Lines indicate the levels obtained for analysis. (B) Cross section of Level 1. Region 1 corresponds to mucociliary epithelium and region 2 to the transitional epithelium lining the lateral surface of the nasoturbinate. NT, nasoturbinate; DM, dorsal medial meatus; S, septum. (C) Cross section of Level V. NPM, nasopharyngeal meatus.

 
BrdU immunostaining.
Hydrated tissue slides were immersed in 4M HCl for 20 min at 37°C to denature DNA. The slides were washed in double distilled water and immersed in pepsin solution (DAKO, Carpinteria, CA) for 15 min at 37°C to expose antigens by breaking protein-formalin crosslinks formed during fixation. Incubation of slides in a solution of pepsin prior to immunostaining with BrdU greatly enhanced the visualization of labeled cells. The slides were rinsed with distilled water followed by rinsing in 10% polyethylene-sorbitan monolaurate (Tween 20) in PBS. Tissue sections were stained using a monoclonal antibody to BrdU from DAKO (Carpinteria, CA) and the DAKO Envision System Peroxidase Kit for detection of the antigen-antibody complex. BrdU incorporation was visualized by final incubation with the chromogen 3,3'-diaminobenzidine tetrahydrochloride (DAB) and DAB Enhancer (Innovex Bioscienes, Richmond, CA). Slides were counterstained with hematoxylin. BrdU labeled cells were recognized by the brown pigmentation of the nuclei.

Histopathology.
Nasal tissues were examined from animals after 3 or 20 days of exposure. Two levels, level I and level V, were examined from each animal for the present study. Level I is immediately posterior to the upper incisor teeth through the naso- and maxilloturbinates. This level included nasal stratified squamous and respiratory epithelium. This level comprises the specific region where nasal tumors appeared during the cancer bioassays in F344 rats. Level V was posterior to the middle of the first upper molar teeth through the ethmoturbinates and included olfactory epithelium as well as the NRE lining the NPM. The purpose of examining this level was to observe differences in exposure effects in the NRE between the anterior nasal passage at level I and the NPM at level V where all air passing through the rat nasal passages merges. Nasal epithelial lesions were recorded on the basis of functional (type of epithelia) and anatomical location (specific location within the tissue level). The lesions were scored as focal (occurring as small lesions within a region in the epithelium), or as extensive (covering large areas within a section). The grading system consisted of five scores: 0, none; 1, minimal; 2, mild; 3, moderate; and 4, marked. Sections were examined for the following lesions: edema, degeneration of olfactory or respiratory epithelia, rhinitis, erosion of the turbinate epithelium, squamous metaplasia, epithelial hyperplasia, mucous cell hypertrophy or hyperplasia, changes in mucous secretion, and presence of apoptotic bodies. Slides were coded for histopathological examination and read blind, so that the examiner had no knowledge of the exposure group or duration of exposure.

Evaluation of the severity of the mucous cell nests.
The severity index for each exposure group was obtained by adding the severity index for each animal in each exposure group and then dividing by the total number of animals examined per group.

Severity index: {Sigma}(individual severity grades)/{Sigma}(animals examined per group).

Evaluation of BrdU incorporation.
The specific anatomical regions within level I that were analyzed for BrdU incorporation were selected based on histopathology results and on the location of tumors in cancer bioassays. Tumors occurred at the margin of the transitional epithelium in the ventral lateral surface of the nasoturbinate. BrdU incorporation at different PO concentrations and lengths of exposure was examined in the nasal mucociliary epithelium lining the region that comprises the middle septum, the dorsal medial meatus and the medial surface of the nasoturbinates (Fig. 1BGo—region 1) as well as in the transitional epithelium lining the lateral surface of the nasoturbinate (Fig. 1BGo—region 2). Measurements were done on one side of the nasal cavity. Also, the NRE lining the NPM (Fig. 1CGo) was examined to compare changes in cell proliferation between the anterior and posterior regions of the nasal passages after PO exposure. Areas of the NPM close to lymphatic vessels were not counted to avoid interference from existing inflammatory cells. The Unit Length Labeling Index (ULLI; labeled basal cells per mm of basement membrane) was used to quantify BrdU incorporation in the NRE (mucociliary and transitional) during S-phase. The ULLI was determined by dividing the total number of BrdU stained cells in each of the regions by the corresponding length of the basement membrane of that region. About 25 fields (~150 µm basement membrane length) were evaluated in region 1. For region 2, about 12 fields (~150 µm) were counted. In the NRE, exposure effects such as cell loss or hyperplasia could affect the total cell number of the surface of the epithelia. ULLI measurements of the NRE are not affected by changes in total cell number, as it measures the length of the basement membrane. The Labeling Index (% LI; labeled cells per total amount of cells) was utilized for the quantitation of BrdU labeled cells in liver. Enough sections were evaluated to count a total of 1200 hepatocytes (labeled and nonlabeled) per liver. Randomly selected fields in the median and left lobes were evaluated. The slides were examined without knowledge of exposure group or time of exposure.

Data analysis.
Ordinary ANOVA was used to test for the effects of PO exposure on cell replication (BrdU incorporation) in nasal and hepatic tissue. Significant differences between control and exposed animals were evaluated with a post hoc comparison procedure (Dunnett’s test). The level of statistical significance was set at p <= 0.05. All reported p’s are two-sided. Statistical analyses were done with a commercial statistical analysis package (SYSTAT 5.05; SPSS, Inc.).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Histopathology
Histopathological examination of selected levels in the nose revealed mainly one treatment-associated lesion in level I. The observed lesions were mainly associated with mucous cells and were characterized by the appearance of multiple glandular-like structures, nests of mucous cells, in the NRE of the medial septum, dorsal medial meatus and medial surface of the NT (Region 1) of the nose of rats exposed to the compound for 20 days (300 or 500 ppm) (Fig. 2Go). In most cases, these lesions caused disarrangement of the epithelium. These lesions were classified as minimal or mild. They were distributed along the region comprising the medial septum, dorsal medial meatus and medial surface of the NT only. A small degree of cell disarrangement is normal in the dorsal medial meatus due to the presence of ducts from seromucous glands (Monticello et al., 1990Go). In fact, these lesions were observed in controls and in animals exposed to low concentrations of PO (<=50 ppm), but they were smaller in size, fewer in number, and were confined to the dorsal medial meatus. The number of glandular-like lesions observed in exposed animals was about eight times greater than in controls. The incidences of mucous cell nests in the NRE lining the anterior nasal passages at Level I at different concentrations and lengths of exposure are shown in Table 1Go. The values reported in the table were obtained by counting the total number of mucous cell nests in the medial septum, dorsal medial meatus and medial surface of the nasoturbinate in both sides of the nasal cavity for each rat, and dividing by the number of rats examined per group. The severity index of the mucous cell nests is summarized in Table 2Go. Exposure of rats to PO for 3 days did not result in an increase in mucous cell nests. The presence of mucous cell nests was not observed in the epithelial lining of the nasopharyngeal meatus of control or exposed rats (Level V). Apoptotic bodies were not observed at any concentration or length of exposure. Necrosis and cell debris was minimal to absent in the exposed groups, but flushing of the nasal cavity during fixation could have washed away cell debris present in the lumen and on the surface of the epithelium lining the affected areas.



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FIG. 2. Photomicrographs of rat nasal respiratory epithelial (NRE) cells (level I) from the dorsal medial meatus stained with hematoxylin and eosin (H&E). Panel A corresponds to a control animal. Normal size mucous cells are identified with an arrow. Panel B corresponds to a rat exposed to 300 ppm PO for 20 days. Nest-like cell infolds, identified with an arrow, cover most of the area. Magnification x200.

 

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TABLE 1 Average Number of Mucous Cell Nests Lining the Dorsal Medial Meatus, Medial Septum, and Medial Surface of Nasoturbinates of F344 Rats Exposed to PO by Inhalation for 3 or 20 Days
 

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TABLE 2 Severity of Mucous Cell Lesions in the Anterior Nasal Passages of Rats Exposed to PO for 20 Days
 
Evaluation of BrdU Incorporation
Systemic delivery of BrdU was observed in all animals as demonstrated by positive staining of the duodenum in all the tissue blocks. The regions (within Level I) examined for the quantitation of BrdU incorporation were selected based on the histopathological findings and, again, on knowledge of the specific location of tumors in the cancer bioassay (NTP, 1985Go). The nasal passages of rats at level I are characterized by three types of respiratory epithelia (Boorman et al., 1990Go). The ventral surface is lined by stratified squamous epithelium. The NRE lining the septum, dorsal medial meatus, and medial surface of the nasoturbinate is highly ciliated and contains numerous mucous cells. This epithelium is composed of ciliated and nonciliated columnar cells, mucous cells, brush cells, and basal cells. The basal cells differentiate into the distinctive mature cells of this type of epithelium. The epithelium lining the lateral surface of the nasoturbinate is nonciliated or sparsely ciliated and it is composed of cuboidal cells. For the purpose of this study, the respiratory epithelium characterized by the presence of ciliated and nonciliated cells, mucous cells, brush cells and basal cells will be referred to as mucociliary epithelium. The epithelium composed of cuboidal cells will be referred to as transitional epithelium.

The nasal tumors in the NTP study (NTP, 1985Go) were located at the ventral lateral surface of the nasoturbinate according to our histopathological examination. This area includes the boundary between transitional and mucociliary epithelium at Level I. The regions examined for BrdU incorporation were region 1 (region comprising the mucociliary epithelium lining the medial septum, dorsal medial meatus and medial surface of the NT) and region 2 (region containing the transitional epithelium lining the lateral surface of the NT; Fig. 1BGo). Region 1 represents those areas where lesions (mucous cell nests) were observed during histopathological examination of tissue slides. Region 2 comprised the section of the margin of the NT and part of the lateral surface of the NT that are lined by transitional epithelium.

A 3.6- and 6.9-fold significant increase in BrdU incorporation above control was observed in region 1 (medial septum, dorsal medial meatus, and medial surface of NT) after exposure of rats to 300 and 500 ppm for 3 days, respectively (Fig. 3AGo). Exposure of rats for 20 days resulted in a 4.4- and 5.4-fold increase in cell proliferation in region 1 at 300 and 500 ppm, respectively. Based on these numbers, the increase in cell proliferation was sustained over the entire exposure period of four weeks. There were no treatment-related increases in cell proliferation at low concentrations of PO (5–50 ppm) at either length of exposure in this region.



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FIG. 3. Cell proliferation measurements in respiratory epithelium lining the septum, dorsal medial meatus, and medial surface of the nasoturbinates in the anterior nasal passages (A), lateral surface of NT (B), respiratory epithelium lining the nasopharyngeal meatus (C), and liver (D) of male F344 rats exposed to PO by inhalation for 3 or 20 days. *Significantly different from control, p < 0.01.

 
A significant increase in BrdU incorporation in region 2 was observed at 500 ppm (4.3-fold) after 3 days of exposure. Significant increases in region 2 cell proliferation after 20 days of exposure to 300 and 500 ppm PO were 2.3- and 3.3-fold from control, respectively (Fig. 3BGo). Changes in cell proliferation compared to control were not observed in region 2 after exposure to low concentrations (5–50 ppm) of PO.

A significant increase in cell proliferation in the epithelial lining of the NPM was evident only at 500 ppm for the 3-day exposure (3.4-fold increase). Exposure to the compound for 20 days led to significant increases in cell proliferation in the NPM at 300 (2.8-fold) and 500 ppm (3.3-fold; Fig. 3CGo). The fold increase in cell proliferation in the mucociliary region lining the NPM was smaller than for the same type of epithelium lining region 1. Inhalation exposure of F344 rats to PO did not have an effect on cell replication in the liver at any concentration or length of exposure (Fig. 3DGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell replication plays a key role at each stage in the evolution of cancer (Croy, 1993Go; Swenberg, 1993Go). Endogenous or chemically induced DNA damage can be converted into mutations during cell replication. Even though the cell has efficient DNA repair systems, an increase in cell proliferation decreases the time available for repair of DNA damage. Thus, chemicals that induce DNA damage and/or increase the rate of cell replication also increase the probability for occurrence of genetic changes that lead to neoplastic transformation.

In the current standard approach for cancer risk assessment, the conservative assumption that cancer incidence decreases linearly with decreasing dose throughout all possible exposure concentrations is a standard default. This approach does not utilize mechanistic data that may be available for the chemical being assessed. Tumor data from rodent bioassays obtained at concentrations significantly larger than those to which humans are exposed are often used to predict risk at low exposure concentrations.

There are an increasing number of examples of nonlinear dose response relationships where the slope of the tumor incidence curve was found to change drastically at high doses. For formaldehyde, the appearance of nasal tumors at high exposure concentrations during the rodent bioassays was associated with an increase in compensatory cell proliferation in the target tissue (Monticello et al., 1996Go). No significant increases in cell proliferation were observed at low concentrations that did not result in nasal tumor formation. This observation provides evidence that sustained increases in cell replication may play a role in determining the slope of the tumor incidence curve. It also suggests that chemicals eliciting a neoplastic response in the presence of increased cell proliferation may pose an insignificant risk for humans exposed to concentrations where changes in the cell proliferation rate were not observed in animal studies. In this context, cell proliferation is a piece of mechanistic data that in conjunction with information on other biological parameters such as tissue dosimetry, toxicokinetics, and detoxication would improve the accuracy of the cancer risk assessment process.

This study demonstrated that exposure of rats to high concentrations of PO (>=300 ppm) for 20 days results in the appearance of hyperplastic lesions (mucous cell nests) in the NRE lining the septum, medial and dorsal surfaces at level I. These histopathological changes in the sections examined were only observed in animals exposed to concentrations at or above 200 ppm. Exposure to concentrations below 300 ppm for 20 days did not result in histopathological changes in the nasal sections examined. The hyperplastic response was associated with mucous secretory cells. The mucous cells were arranged in clusters or mucous cell nests. The morphological appearance of the lesions observed in this study is comparable to observations from previous short-term studies performed by Eldridge et al.(1995)Go and to the cancer bioassays results (Lynch et al., 1984bGo; NTP, 1985Go; Renne et al., 1986Go). These lesions did not increase in number over controls in rats exposed to low concentrations (<=50 ppm) or in groups of rats exposed to PO for 3 days. Nasal mucous cell hyperplasia is a common response to mucosal injury (Boorman et al., 1990Go). It has been observed in rodents exposed to gaseous irritants such as formaldehyde (Chang et al., 1983Go; Monteiro-Riviere and Popp, 1986Go; Swenberg et al., 1983Go) and glutaraldehyde (St. Clair et al., 1990Go).

The region within level I that was characterized by PO-induced mucous cell hyperplasia based on the histopathology results (region 1) correlated with the region with the largest increase in BrdU incorporation at high exposure concentrations. Exposure of rats to PO for 3 days led to a 3.6- and 6.9-fold increase in cell proliferation (region 1) in the groups of rats exposed to 300 or 500 ppm, respectively. After 20 days of exposure to 300 and 500 ppm PO, increases in cell proliferation over controls were 4.4- and 5.4-fold, respectively. The increase in cell replication remained sustained after 20 days of exposure. The concentrations at which significant increases in cell proliferation were observed in region 1 correlate with those eliciting a histopathological response in this region. As previously demonstrated by Eldridge et al.(1995)Go, the increase in cell proliferation as well as the increased tissue damage in region 1 were reversible. The removal of the insult, in this case PO exposure, permits tissue repair. The increase number of mucous cell nests (region 1) could be the result of basal cell replication leading to selective mucous cell differentiation. The increase in mucous secretory cells is most likely an adaptive response to repeated insult, as it was only observed after longer times of exposure (20 days). The increased mucous secretion in this area may offer a degree of protection to this region. In the present study, a no-observed-effect level (NOEL) for increased cell proliferation and tissue damage in the mucociliary epithelium (region 1) after exposure of rats for 20 days was established at 50 ppm. This is in accordance with observations from a previous short-term PO inhalation study in rats (Eldridge et al., 1995Go).

The location of the nasal tumors in rats exposed to high concentrations of PO for their lifetime was found at the ventral lateral surface of the NT. The boundary between the mucociliary and the transitional epithelial is found in this area. No histopathological changes were observed in region 2 (transitional epithelium lining lateral surface of nasoturbinate) at any concentration or length of exposure. However, examination of BrdU incorporation in rats exposed to PO for 3 days demonstrated a statistically significant increase in cell replication in the transitional epithelium lining the lateral surface of the nasoturbinate (region 2) at 500 ppm PO (4.3-fold). Also, measurements of BrdU incorporation in region 2 of rats exposed to 300 and 500 ppm for 20 days led to 2.3- and 3.3-fold differences from control respectively. This is the first time that cell proliferation measurements have been obtained for the transitional epithelium lining the lateral surface of the nasoturbinates (region 2) after PO inhalation exposure. There is no information available on whether or not the increase in cell proliferation in this region is reversible after cessation of PO exposure. In contrast to the mucociliary epithelium, the transitional epithelium is characterized by a low number of mucous secretory cells. This type of epithelium may be more susceptible to repeated insult in view of the minimal amount of mucous secreting cells in the epithelium. Although the histopathological evaluation did not show evidence of tissue damage, we believe that the increase in cell proliferation in region 2 is a response to cytotoxicity, which is only observed at high exposure concentrations. This increase in cell proliferation is likely to be related to the induction of tumors in this area. No significant increases in cell proliferation above control were observed in region 2 of animals exposed to concentrations of 300 ppm or below after 3 days of exposure. A NOEL for increased cell proliferation in region 2 after 20 days of exposure to PO was established at 50 ppm.

The difference in susceptibility between the two types of nasal respiratory epithelium, mucociliary and transitional, was not well understood prior to this experiment. It is known that the different types of epithelium respond or adapt differently to tissue insult. Mucous cell hypertrophy and hyperplasia occur as an adaptive response to tissue insult in the nasal mucociliary epithelium (region 1). Squamous metaplasia and epithelial cell proliferation are usually observed as responses in the nasal transitional epithelium (Boorman et al., 1990Go; Harkema, 1990Go). Knowledge of differences in tissue dosimetry between these two regions would help determine how much of the compound each region receives and thus their relative susceptibility to insult. The results of this study suggest that PO is not highly cytotoxic, as such toxic lesions were not observed. On the other hand, region 2 is less well protected by mucociliary flow than region 1 and did not gain additional protection by the adaptive goblet cell hyperplasia that occurred in region 1. Thus, it is possible that the molecular dose of PO reaching the epithelium in region 2 was somewhat greater. This, coupled with an increase in cell proliferation, is the most likely reason for the selective localization of the PO-induced nasal adenomas. As such, the cell proliferation data for region 2 represents a key event in the mode of action of PO-induced carcinogenesis.

Changes in cell proliferation were also observed in the nasal mucociliary epithelium lining the nasopharyngeal meatus (NPM) in level V. As with the anterior nasal passages, significant changes in cell replication were only observed at carcinogenic concentrations. A 3.4-fold increase from control was shown after exposure of rats to 500 ppm PO for 3 days. Exposure for 20 days resulted in a 2.8- and a 3.3-fold increase in rats exposed to 300 and 500 ppm, respectively. The milder effect of PO on the posterior part of the nose could be related to less of the compound reaching this site, either by being deposited in the anterior portion of the nose and/or better detoxication in this area. There was no evidence of tumor formation in the area corresponding to level V (including NPM) in the cancer bioassays.

The results from this study in conjunction with observations from a previous short-term study (Eldridge et al., 1995Go) identified a nonlinear increase in cell proliferation in the nasal mucociliary epithelium after PO exposure with a threshold concentration around 150 ppm. A nonlinear exposure concentration response was also found for hyperplastic lesions with a lower threshold value (50 ppm).

In companion studies of the present research, DNA adducts (7-HPG) were present in the nasal respiratory epithelium at concentrations ranging from 5 to 500 ppm. Figure 4Go compares the exposure response relationship between nasal adenomas, DNA adducts, and cell proliferation in rats exposed by inhalation to PO. While the exposure response is clearly nonlinear for nasal adenomas, the exposure response for 7-HPG was linear from concentrations that induced nasal adenomas (300–500 ppm) to those that were shown to be noncarcinogenic in rodent bioassays (<=200 ppm; Ríos-Blanco et al., in press). As shown in this study, the exposure concentrations ranging from 5 to 50 ppm PO did not induce nasal tissue damage or increases in nasal respiratory cell proliferation. When the present data are combined with that of Eldridge et al.(1995)Go, there is a clear nonlinear response for cell proliferation that correlates well with that of nasal adenomas. These observations suggest that the formation and accumulation of 7-HPG adducts in the nasal mucosa are not a sufficient factor in PO toxicity or carcinogenesis to drive the exposure response. In contrast, PO-induced cell proliferation is only present at carcinogenic concentrations of PO (>=300 ppm). Together, the data suggest that cell proliferation is a critical factor for tumorigenesis in this tissue.



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FIG. 4. Comparison of nasal adenoma incidence (NTP, 1985Go), nasal respiratory epithelium 7-hydroxypropylguanine DNA adducts (Rios-Blanco et al., in press) and cell proliferation data from the present study and that of Eldridge et al.(1995)Go. The cell proliferation data is presented as fold increase over controls, with Eldridge et al. data points at 0, 50, 150 and 525 ppm PO and the present data at 0, 5, 20, 50, 300, and 500 ppm PO for 20 days.

 
Exposure of rats to PO for 3 or 20 days showed no effect on cell replication in the liver, a nontarget tissue for PO carcinogenesis. The absence of an effect on the cell proliferation rate in liver could be related to a less of the compound being distributed systemically. Previous studies (Ríos-Blanco et al., 1997Go, 2000Go; Segerbäck et al., 1998Go) and the new dosimetry study (Ríos-Blanco et al., in press) demonstrated a much higher level of DNA alkylation in the nose than in systemic organs after inhalation exposure to 500 ppm PO for 20 days. Thus, it is not surprising that no cytotoxic or proliferative responses were present.

PO is a direct DNA alkylating agent, but a weak mutagen. The presence of DNA damage at exposure concentrations that have been shown to induce a significant increase in cell replication may augment the chance of developing mutations. Alternatively, the probability of developing a mutation at lower levels of exposure where biologically significant increases in cell proliferation are not present is small. This is especially true for the nasal respiratory epithelium, which is characterized by a low cell turnover rate (0.5%; Shorter et al., 1966Go).

BrdU pulse labeling techniques were utilized in previous studies on PO-induced nasal cell proliferation (Eldridge et al., 1995Go). The current study used osmotic pumps for the delivery of BrdU to tissues. Osmotic pumps offer greater sensitivity than pulse-labeling techniques due to a longer labeling time and efficient delivery of the thymidine analog to tissues. For instance, an increase (4X) in sensitivity was observed over previous studies conducted by BrdU pulse labeling techniques in the region corresponding to the mucociliary epithelium (region 1).

In summary, significant increases in cell proliferation in the nasal mucociliary and transitional epithelia were only observed at concentrations that were carcinogenic in the rodent bioassays. Histopathological changes were only observed at concentrations found to be carcinogenic in rats. Significant increases in cell proliferation were identified in regions where tumors formed in the rodent bioassay. A NOAEL for hyperplastic changes in the mucociliary epithelium (region 1) was identified at 50 ppm. In this study, a NOEL for cell proliferation in the mucociliary epithelium was observed at 50 ppm, however, previous studies have demonstrated that inhalation to 150 ppm PO for 20 days does not lead to a significant increase in cell replication from control. Therefore, a NOEL of 150 ppm can be established for the cell proliferation response in rats exposed to PO by inhalation.

Data from this study, along with data on tissue dosimetry and detoxication, provides important information related to the mode of action for PO carcinogenesis. While the molecular dose of DNA damage was linear, cell proliferation in the transitional epithelium of the nasal turbinate was nonlinear and correlated best with tumor induction. Use of these data will improve the accuracy of PO cancer risk assessments.


    ACKNOWLEDGMENTS
 
The authors greatly appreciate the help of members from Dr. Johannes Filser’s research group at the GSF Institute of Toxicology. The authors will also like to thank Dr. Judith Baldwin for quality assurance of the data, and Teresa Bone and Janice Weber for preparation of excellent histology and immunohistochemistry slides.

This research was supported in part by the Propylene Oxide/Propylene Glycol Panel and the Olefins Panel of the American Chemistry Council, by the Propylene Oxide and Derivatives Sector Group of the European Chemical Industry Association (CEFIC), and NIH grants T32 ES07126, P30 ES10126, and P30 CA16086. The authors made all decisions on interpretation and wording of the article.


    NOTES
 
1 To whom correspondence should be addressed at Department of Environmental Sciences and Engineering, Rosenau Hall 253c, CB# 7431, Chapel Hill, NC 27599. Fax: (919) 966-6123. E-mail: james_swenberg{at}unc.edu. Back


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