Lung Tumor Induction by Inhalation Exposure to Molybdenum Trioxide in Rats and Mice

TOXICOLOGICAL SCIENCES ARTICLE NO.

45, 58 – 65 (1998)

TX982497

Lung Tumor Induction by Inhalation Exposure to Molybdenum Trioxide in Rats and Mice P. C. Chan,* R. A. Herbert,* J. H. Roycroft,* J. K. Haseman,* S. L. Grumbein,† R. A. Miller,† and B. J. Chou† *National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; and †Battelle Pacific Northwest Laboratories, Richland, Washington 99352 Received August 25, 1997; accepted May 19, 1998

and hexavalent states. The biological differences with respect to valence are not clear. Molybdenum trioxide is a hexavalent compound (Kirk-Othmer, 1981; Goyer, 1991) produced by roasting an ore containing molybdenum at 649°C. It is used primarily (90% or more) as an additive to steel and corrosion-resistant alloys. It is also used as a chemical intermediate for molybdenum products; an industrial catalyst; a pigment; a crop nutrient; a component of glass, ceramics, and enamels; a flame retardant for polyester and polyvinyl chloride resins; and a reagent in chemical analyses (Kirk-Othmer, 1981; NIOSH/OSHA, 1981; Clayton and Clayton, 1981). Molybdenum trioxide is water soluble and is absorbed rapidly after inhalation exposure. After temporary retention in the tissues, molybdenum is excreted completely in the form of molybdate, with no accumulation in mammals (Friberg, 1979). Dietary administration of sodium molybdate to rats for periods of 4 –7 weeks induced femurotibial joint enlargement (Miller et al., 1956) and mandibular and maxillary exostoses (Ostrum et al., 1961; Van Reen, 1959). Weanling male rats fed in a containing ammonium tetrathiomolybdate and copper showed severe changes at long bone growth plates and muscle insertions and beneath the periosteum (Spence et al., 1980). Thickening and widening of the epiphyseal growth plates of the femur, tibia, humerus, radius, and ulna were observed. Renal failure and fatty degeneration in kidney and liver have been reported following oral administration of molybdenum salt to rats (USEPA, 1975; Bompart et al., 1990). Molybdenum trioxide was selected for study because of its large production volume, wide industrial use, high potential for human exposure, and lack of toxicological data.

Lung Tumor Induction by Inhalation Exposure to Molybdenum Trioxide in Rats and Mice. Chan, P. C., Herbert, R. A., Roycroft, J. H., Haseman, J. K., Grumbein, S. L., Miller, R. A., and Chou, B. J. (1998). Toxicol. Sci. 45, 58 – 65. Inhalation studies of molybdenum trioxide (MoO3) were conducted because of its wide use in industry, human exposure, and lack of data on carcinogenicity. Groups of 50 male and 50 female F344/N rats and B6C3F1 mice were exposed to MoO3 by inhalation at 0, 10, 30, or 100 mg/m3, 6 h/day, 5 days/week, for 2 years. In both rats and mice, survival and mean body weights of exposed groups of males and females were similar to those of their respective controls. There were significant exposure-dependent increases in blood molybdenum concentration in exposed rats and mice. There were no toxicological differences in bone density or curvature between exposed animals and their respective controls. In rats, dose-dependent increases in incidence of hyaline degeneration in the nasal olfactory epithelium and squamous metaplasia of the epithelium lining the base of the epiglottis were observed. The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) was marginally increased in males but not in females compared with controls. In mice, the incidences of squamous metaplasia of the epithelium lining the base of the epiglottis, hyperplasia of the laryngeal epithelium, and metaplasia of the alveolar epithelium were significantly increased in all exposed males and females compared with controls. The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) in exposed groups of males and females was significantly greater than that in the control groups. © 1998 Society of Toxicology.

Molybdenum is an essential element in plants and animals. The metal is required for the function of the nitrogenfixing enzyme, nitrogenase, in nitrogen-fixing bacteria, and in plants. It is a cofactor for the enzymes xanthine oxidoreductase, sulfide oxidase, and aldehyde oxidase in mammals (USEPA, 1975). Molybdenum is widely distributed in nature and is present in fossil fuels such as coal and oil; in sea, river, and drinking water; and in city air (Hammond and Beliles, 1980). Molybdenum occurs in five valence states, 12, 13, 14, 15, and 16, and forms two series of stable and water-soluble salts in tri1096-6080/98 $25.00 Copyright © 1998 by the Society of Toxicology. All rights of reproduction in any form reserved.

MATERIALS AND METHODS Chemical. Molybdenum trioxide was obtained from S. W. Shattuck Chemical Co., Inc. (Houston, TX) and Climax Molybdenum Co. (Greenwich, CT). The purity of the compound was determined by elemental analyses including atomic absorption spectroscopy, gravimetric analysis, Karl Fischer water analysis, spark source mass spectrometry, and inductively coupled plasma atomic emission spectrometry. The overall purity of the compound was 99%. Details of the analyses were described in the National Toxicology Program (NTP) Technical Report No. 462 (1996). 58

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MOLYBDENUM TRIOXIDE CARCINOGENESIS Animals. Groups of 50 male and 50 female F344/N rats and B6C3F1 mice (6 weeks old), obtained from Simonsen Laboratories (Gilroy, CA) were exposed to molybdenum trioxide by inhalation in chambers at concentrations of 0, 10, 30, or 100 mg/m3, 6 h per day, 5 days per week, for 104 (mice) or 105 (rats) weeks. The exposure concentrations were selected based on results of 13-week studies in which rats and mice were exposed at up to 100 mg/m3 with no effects on mortality and body weight (NTP, 1996). The animals were housed individually. Water was available ad libitum. Feed was also available ad libitum except during exposure periods. Cages and racks were rotated weekly. All animals were observed twice daily. Body weights were recorded weekly for the first 12 weeks, at week 15, monthly thereafter, and at the end of the studies. Clinical observations were recorded initially; at 4, 8, 12, and 15 weeks; monthly thereafter; and at the end of the studies. At the end of the study a complete necropsy was performed on all rats and mice. At necropsy, all organs and tissues were examined for grossly visible lesions, and all selected tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 mm, and stained with hematoxylin and eosin for microscopic examination. Blood was collected at the end of the studies and blood molybdenum concentrations were determined with inductively coupled plasma atomic emission spectrometry. Bone density was calculated by weighing freshly collected right femurs both in air and suspended in water. Femoral curvature was assessed by measuring two lengths defined between points on the femur tangential to a straight line placed along the medial aspect. The curvature was defined as the ratio of the length following the outline of the bone and the straight-line distance between the landmarks. Inhalation system. The molybdenum trioxide aerosol generation and delivery systems were composed of four basic components: a flexible-brush dust-feed mechanism, a Trost air impact mill, an aerosol charge neutralizer, and an aerosol distribution system. The Trost air impact pulverlizer used the fluid energy from opposing air jets to cause particle-to-particle, head-on impaction to deagglomerate and reduce the size distribution of the material. Aerosol passed through the charge neutralizer and through the distribution line. At each chamber location, an Air-Vac pump withdrew material from the distribution line into the chamber inlet. Each distribution line branch terminated with a high-efficiency particulate air (HEPA) filter to remove any excess material. During the study, chamber molybdenum trioxide aerosol concentration was monitored using a real-time aerosol monitor (RAM; MIE, Inc., Bedford, MA). Readings were recorded approximately hourly for each chamber and were used to make adjustments to the dust generating systems. RAMs were calibrated twice monthly. Samples were taken daily from closed-face Gelman DM-450 Metricel filters in each exposure chamber for gravimetric analysis of chamber concentration to verify the RAM calibration. Particle size distributions in each chamber were determined using an Anderson eight-stage cascade impactor with an 11-mm preseparator. Impactor (Mercer-style seven-stage impactor; In-Tox Products, Albuquerque, NM) samples were taken from each exposure chamber at monthly intervals. The mass median aerodynamic particle diameter and the geometric standard deviation were estimated to range from 1.3 to 1.8 mm. Uniformity of aerosol concentration in the exposure chambers was evaluated every 3 months from 12 chamber positions. The means of concentration in all chambers were at least 95% of the target. Details of analyses were described in the NTP Technical Report No. 462 (NTP, 1996). Statistical analysis. Differences in survival were analyzed by life table methods (Cox, 1972). Tumor incidence data were analyzed by survivaladjusted methods (Haseman, 1984; NTP, 1996) and by Fisher exact tests and Cochran-Armitage trend tests based on the overall proportion of tumor-bearing animals (Gart et al., 1979). P values reported for tumor comparisons are one-sided. Details of the experimental procedures and data evaluations can be found in NTP Technical Report No. 462 (NTP, 1996).

TABLE 1 Blood Molybdenum Concentration in Rats in the 2-Year Study

Male n Mo (mg/g) Female n Mo (mg/g) a

0 mg/m3

10 mg/m3

30 mg/m3

100 mg/m3

8a 0.22 6 0.05

10 0.80 6 0.12

10 1.77 6 0.30

10 6.04 6 1.10

6a 0.06 6 0.01

10 0.36 6 0.07

10 0.66 6 0.10

10 2.41 6 0.48

Some of the measurements were below the limit of detection (0.03 mg/g).

RESULTS

Rat Studies Survival of exposed males and females was similar to that of controls, averaging 29% for males and 47% for females. Mean body weights of exposed groups of males and females were similar to controls throughout the study. No clinical findings related to molybdenum trioxide exposure were observed. There was a significant dose-dependent increase in blood molybdenum concentration in exposed rats (Table 1). Blood concentrations of molybdenum in exposed males were greater than those in exposed females. There were no toxicologically significant differences in bone density or curvature between control and exposed rats. The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) was increased in male rats with a marginally significant (P 5 0.034) positive trend (Table 2). No increase in the incidence of pulmonary neoplasms occurred in female rats. The incidence of chronic inflammation in the lungs of male and female rats exposed to 30 or 100 mg/m3 was significantly greater than that in controls. The severity of chronic inflammation was greater in rats exposed to 100 mg/m3 compared to control rats or rats exposed to 10 or 30 mg/m3. Chronic inflammation in the lungs of control rats consisted of focal intraalveolar aggregates of foamy macrophages. Chronic inflammation in the lungs of exposed rats was a multifocal lesion localized at peribronchiolar and subpleural sites (Figs. 1 and 2). Lesions were variably sized, sharply delineated, and densely cellular, consisting of large foamy macrophages, with lesser numbers of epithelioid cells, multinucleated giant cells, and neutrophils mixed with cellular debris (Fig. 3). Cholesterol crystals (clefts) within macrophages, multinucleated giant cells, and free among the inflammatory cells were present in many lesions (Fig. 4). Within lesions, alveolar septae were thickened by mild interstitial fibrosis, infiltrates of mononuclear inflammatory cells, and hyperplastic type II epithelial cells. Alveoli in some advanced lesions were lined by ciliated cuboidal or columnar cells (metaplasia) and frequently contained mucus. In the most severe lesions, fibrous tissue obliterated the alveolar architecture. Incidence of hyaline degeneration in the nasal respiratory

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TABLE 2 Incidences of Respiratory System Lesions in Male and Female Rats Exposed to MoO3

Male Lung Alveolus chronic inflammation Alveolar/bronchiolar adenoma Carcinoma Ad/Ca Nose Respiratory epithelium hyaline degeneration Larynx Epiglottis squamous metaplasia Female Lung Alveolus chronic inflammation Alveolar/bronchiolar adenoma Carcinoma Ad/Ca Squamous cell carcinoma Nose Olfactory epithelium hyaline degeneration Respiratory epithelium hyaline degeneration Larynx Epiglottis squamous metaplasia

0 mg/m3

10 mg/m3

30 mg/m3

100 mg/m3

2/50 0/50 0/50 0/50

3/50 0/50 1/50 1/50

25/50** 0/50 1/50 1/50

47/50** 3/50 1/50 4/50

2/50

7/49

48/49**

49/50**

0/49

11/48**

16/49**

39/49**

14/50 0/50 0/50 0/50 1/50

13/50 1/50 1/50 2/50 0/50

43/50** 0/50 0/50 0/50 0/50

49/50** 2/50 0/50 2/50 0/50

39/48 1/48

47/49* 13/49**

50/50** 50/50**

50/50** 50/50**

0/49

18/49**

29/49**

49/50**

* P , 0.05. ** P , 0.01.

epithelium in 30 and 100 mg/m3 males and in all exposed groups of females was significantly greater than those in the control groups (Table 2). The incidence of hyaline degeneration in the nasal olfactory epithelium of all exposed groups of females was significantly greater than that in the control group (Table 2). Hyaline degeneration generally affected the respiratory epithelium of the nasal septum in level II of the nasal cavity and

FIG. 1. Peribronchiolar chronic inflammation in the lung of a female rat exposed to 100 mg/m3 molybdenum trioxide by inhalation for 2 years. TB, terminal bronchiole. H&E; 453.

the olfactory epithelium in levels II and III. The epithelium in these regions contained variably sized brightly eosinophilic cytoplasmic globules which often filled and distorted the cells (Fig. 5). Affected segments of the olfactory epithelium often had fewer cell layers than normal and disorganization of the typically layered rows of nuclei. Neoplasms of the nasal cavity were not observed. Incidence of squamous metaplasia of the epithelium lining the base of the epiglottis in all exposed groups of male and

FIG. 2. Subpleural chronic inflammation in the lung of a male rat exposed to 30 mg/m3 molybdenum trioxide by inhalation for 2 years. H&E; 363.

MOLYBDENUM TRIOXIDE CARCINOGENESIS

FIG. 3. Peribronchiolar chronic inflammation in the lung of a male rat exposed to 100 mg/m3 molybdenum trioxide by inhalation for 2 years. Alveoli contain aggregates of large foamy macrophages (*). Alveolar septae are lined by cuboidal hyperplastic type II cells (arrows). TB, terminal bronchiole. H&E; 1803.

female rats was significantly greater than those in controls and increased with increasing exposure concentration (Table 2). In affected rats, the single layer of ciliated cuboidal to columnar epithelium that normally lines the base of the epiglottis (Fig. 6) was replaced by a basal layer of cuboidal cells with one or more superficial layers of flattened epithelial (squamous) cells that lacked cilia (Fig. 7). Neoplasms of the larynx were not observed. Mouse Studies Survival rates of exposed male and female groups were similar to those of control groups, averaging 63% for males and 66% for females. Mean body weights of exposed males were similar to and those of females were slightly greater than

FIG. 4. Chronic inflammation in the lung of a female rat exposed to 100 mg/m3 molybdenum trioxide by inhalation for 2 years. Note macrophages within alveoli and alveolar septa, type II cell hyperplasia and cholesterol clefts (arrowheads). An alveolus contains aggregates of neutrophils mixed with cellular debris (asterisk). H&E; 1503.

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FIG. 5. Hyaline degeneration of the olfactory epithelium in Level III of the nasal cavity of a male rat exposed to 100 mg/m3 molybdenum trioxide by inhalation for 2 years. Large eosinophilic globules (arrows) fill the cytoplasm of epithelial cells of the ethmoid turbinates. The epithelium has fewer cell layers and there is disorganization of the normally layered rows of sensory cell nuclei. H&E; 1803.

those of controls. No clinical findings related to molybdenum trioxide exposure were observed. There was a significant dosedependent increase in blood molybdenum concentration in exposed mice (Table 3). There were no differences in bone density or curvature between control and exposed mice. The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) in all dosed groups of exposed male and female mice was greater than those of the controls (Table 4). The differences were statistically significant (P , 0.05) for low- and mid-dose male and for high-dose female mice. Alveolar adenomas were discrete nodular masses composed of well-differentiated epithelial cells that formed papillary or solid patterns which distorted the underlying architecture and sometimes compressed the adjacent alveolar parenchyma (Fig. 8). Papillary neoplasms were composed of irregular papillary

FIG. 6. Normal ciliated columnar epithelium lining the base of the epiglottis (arrows) of a male rat exposed to 100 mg/m3 molybdenum trioxide by inhalation for 2 years. H&E; 1803.

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FIG. 7. Squamous metaplasia of the epithelium at the base of the epiglottis of a male rat exposed to 100 mg/m molybdenum trioxide by inhalation for 2 years. Flattened (squamous), nonciliated epithelium (arrows) replaces the ciliated columnar epithelium normally present in this location. H&E; 563.

epithelium lining the base of the epiglottis was significantly increased in all exposed groups of males and females (Table 4). Squamous metaplasia was similar to that observed in rats. The normally single layer of pseudostratified ciliated cuboidal to low columnar epithelium lining the base of the epiglottis was replaced by a hypercellular layer of basal cells with one or more superficial layers of flattened (squamous) epithelial cells that lacked cilia. In advanced cases, there was slight keratinization of the epithelial surface. The incidences of hyperplasia of the laryngeal epithelium in level II of the larynx were significantly increased in male and female mice exposed to 100 mg/m3. Hyperplasia was characterized by increased thickness of the stratified squamous epithelium that normally covers the medial and ventral aspects of the corniculate processes of the arytenoid cartilage of the vocal cords. The severity of both squamous metaplasia of the epiglottis and hyperplasia of the laryngeal epithelium was dose related. DISCUSSION

structures lined by uniform cuboidal cells with abundant cytoplasm and round to oval nuclei. Solid neoplasms were composed of sheets of uniform polygonal cells with abundant eosinophilic cytoplasm and round, oval, or polygonal nuclei. Carcinomas were expansive, and at times invasive masses composed of a pleomorphic population of anaplastic cells in papillary or pleomorphic growth patterns (Fig. 9). Mitotic figures were often numerous. The incidence of metaplasia of the alveolar epithelium in the centriacinar region of lung was significantly increased in all exposed groups of males and females (Table 4). Metaplasia was characterized by a change from the flattened epithelial cell type normally lining alveolar ducts and adjacent alveolar septa to low cuboidal cells considered to be either Clara and/or Type II pneumocytes (Fig. 10). The incidence of alveolar histiocytic infiltration in all exposed groups of males was significantly greater than that in the control group. In the nasal cavity, incidences of hyaline degeneration of the respiratory epithelium in 100 mg/m3 males and females and hyaline degeneration of the olfactory epithelium in 100 mg/m3 females were significantly greater than those in the control groups (Table 4). As in rats, hyaline degeneration was considered a nonspecific degenerative response to chronic inhalation of molybdenum trioxide. Hyaline degeneration affected the respiratory epithelium lining the nasal septum in levels I and II and the medial surfaces of the nasoturbinates in level II; it was confined to the olfactory epithelium lining the dorsal meatus of level II. Microscopically, the lesions were morphologically similar to those observed in rats. The incidences of minimal focal suppurative inflammation in the nasal cavity of 30 and 100 mg/m3 males and focal atrophy of the olfactory epithelium of 100 mg/m3 males were slightly greater than those of the control group. It was not clear whether these lesions were related to chemical exposure. In the larynx, the incidence of squamous metaplasia of the

Molybdenum trioxide is water soluble and is well absorbed after inhalation and oral exposure. In guinea pigs exposed by inhalation to molybdenum trioxide at 204.7 mg/m3, 1 h per day, 5 times per week for 5 weeks, molybdenum concentrations in liver, kidney, lung, spleen, and bone were higher compared with controls (Fairhall et al., 1945). In the present studies, blood molybdenum concentrations in rats and mice increased with increasing exposure concentration; male rats exhibited higher blood concentrations and greater variability than female rats. Blood molybdenum concentrations of male rats were higher than those of female rats and male and female mice exposed to the same exposure concentration. This was probably due to the larger lung capacity of male rats and therefore more molybdenum trioxide was inhaled. The doserelated blood concentrations of molybdenum trioxide in rats and mice in the present studies demonstrated that molybdenum trioxide administered via inhalation was well absorbed. Exposure of rats to molybdenum trioxide at 10, 30, or 100 mg/m3 for 2 years had no effects on survival, body weight gain, or clinical signs compared with controls. Exposure to molyb-

TABLE 3 Blood Molybdenum Concentration in Mice in the 2-Year Study 0 mg/m3 Male n Mo (mg/g) Female n Mo (mg/g) a

10 mg/m3

30 mg/m3

100 mg/m3

0a —

6a 0.10 6 0.01

10 0.21 6 0.03

10 0.77 6 0.11

1a 0.04

6a 0.07 6 0.01

10 0.20 6 0.02

10 0.52 6 0.03

Some or all of the measurements were below the limit of detection (0.04 mg/g).

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MOLYBDENUM TRIOXIDE CARCINOGENESIS

TABLE 4 Incidences of Respiratory System Lesions in Male and Female Mice

Male Lung Alveolar epithelium hyperplsia Alveolar/bronchiolar epithelium metaplasia Adenoma Carcinoma Ad/Ca Nose Inflam. suppurative Olf. epith., atrophy Resp. epith. deg. hyaline Larynx Hyperplasia Epiglottis, sq. metaplasia Female Lung Alveolar epith., hyperplasia Alveolar/bronchiolar epith., metaplasia Adenoma Carcinoma Ad/Ca Nose Olf. epith. deg. hyaline Resp. epith. deg. hyaline Larynx Hyperplasia Epiglottis, sq. metaplasia

0 mg/m3

10 mg/m3

30 mg/m3

100 mg/m3

2/50 0/50 9/50 2/50 11/50

1/50 32/50** 14/50 16/50** 27/50**

6/49 36/49** 10/49 14/49** 21/49*

2/50 49/50** 9/50 10/50* 18/50

2/50 3/50 11/50

6/50 5/50 13/50

10/49 3/49 11/49

8/50* 10/50* 41/50**

1/50 0/50

3/49 26/49**

6/48 37/48**

41/50** 49/50**

1/50 2/50 1/50 2/50 3/50

3/50 26/50*8 4/50 2/50 6/50

3/49 39/49** 8/49* 0/49 8/49

6/49 46/49** 9/49* 6/49 15/49**

14/50 23/50

14/49 28/49

36/49** 48/49**

1/50 36/50**

7/49 43/49**

35/50** 49/50**

22/49 26/49 1/49 1/49

* P , 0.05. ** P , 0.01.

denum is known to cause deformities in the joints (USEPA, 1975). In the present studies, no abnormalities of bone curvature, bone density, or the joints were observed. The rats did not

show signs typical of molybdenum toxicity such as diarrhea, alopecia, achromotricia, dermatosis, and anemia. The amount of molybdenum absorbed in the present studies was probably

FIG. 8. Alveolar/bronchiolar adenoma in the lung of a male mouse exposed to 100 mg/m3 molybdenum trioxide by inhalation for 2 years. The alveolar architecture is effaced by uniform, well-differentiated cuboidal neoplastic epithelial cells arranged in a papillary pattern. H&E; 1503.

FIG. 9. Alveolar/bronchiolar carcinoma in the lung of a male mouse exposed to 100 mg/m3 molybdenum trioxide by inhalation for 2 years. Anaplastic epithelial cells in pleomorphic growth patterns efface the alveolar architecture. H&E; 603.

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FIG. 10. Epithelial metaplasia in the centriacinar region of the lung of a female mouse exposed to 100 mg/m3 molybdenum trioxide by inhalation for 2 years. Cuboidal (metaplastic) epithelial cells (arrows) replace the normally flattened epithelial cells lining the alveolar duct (AD) and the adjacent alveolar septae. H&E; 1253.

below the threshold level needed to initiate such toxicity. The bone and joint abnormalities of molybdenum may be more difficult to detect in young adult rats (7– 8 weeks old) when the skeletal system was almost fully developed. Bone and joint abnormalities were demonstrated by Miller et al. (1956), Van Reen (1959), and Lalich et al. (1965) in weanling rats exposed to high molybdenum and low copper levels. The concentration of copper in the diet (4 mg/kg) in the present studies might have protected the animals from molybdenum toxicity. Exposure of male and female rats to molybdenum trioxide by inhalation resulted in the development of respiratory lesions. In the lung, the incidence and severity of chronic alveolar inflammation increased with increasing exposure concentration. In addition, four alveolar/bronchiolar adenomas or carcinomas were present in male rats exposed to 100 mg/m3; none were present in controls. Lung neoplasms were not observed in female rats exposed to molybdenum trioxide. Chronic inflammatory lesions in lungs and increased incidence of pulmonary neoplasms were observed in rats following inhalation exposure to talc (NTP, 1993), nickel oxide (NTP, 1995a), and nickel subsulfide (NTP, 1995b). The distribution of chronic inflammatory lesions in the lungs of rats in this study is similar to that in the talc, nickel oxide, and nickel subsulfide studies; however, only marginally increased incidences of lung neoplasm were observed. Lesions in the nose (hyaline degeneration) and larynx (squamous metaplasia) were considered to be defensive or adaptive responses to chronic inhalation exposure to molybdenum trioxide. Hyaline degeneration is a common age-related lesion in the nasal epithelium of rats. The incidence and severity of this change has been observed to increase proportionally with exposure concentration in inhalation studies of a variety of irritants. The association between exposure to molybdenum trioxide and the development of pulmonary neoplasms was stronger for

mice than for rats. The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) in the exposed groups of male and female mice was higher than that in the controls. Inherent species differences may explain the pulmonary responses associated with molybdenum trioxide exposure in rats and mice. Fischer 344/N rats have a low background incidence of spontaneous pulmonary neoplasms, whereas the spontaneous incidence in B6C3F1 mice is relatively high. Mice may be more sensitive than rats to pulmonary tumor induction. However, unlike in rats, chronic inflammatory lesions were not present in the lungs of mice. The nasal and laryngeal lesions in mice were similar to those in rats and were considered to be defensive or adaptive responses to chronic inhalation exposure to molybdenum trioxide. The pathogenesis of pulmonary alveolar bronchiolar neoplasms is thought to involve a morphologic and biologic continuum from hyperplasia to carcinoma. Centriacinar alveolar epithelial metaplasia (alveolar bronchiolization, alveolar/bronchiolar hyperplasia) is considered to be a part of the continuum of lesions leading to the development of alveolar/bronchiolar neoplasms after inhalation of particulates. In the present studies, there was a concentration dependent increase in the incidences of alveolar epithelial metaplasia in the centriacinar region of the lungs of all treated groups of male and female mice. However, the severity of this lesion was minimal and did not increase with increasing exposure concentration. In the present studies, the significance of this lesion to the development of alveolar/bronchiolar neoplasms is not clear. Molybdenum trioxide is not mutagenic. The mechanism of action of molybdenum trioxide in pulmonary carcinogenesis is not known. The nonneoplastic lesions observed in the nose and larynx of rats and in the nose, larynx, and lung of mice were apparently attempts by the rats and mice to develop a more durable epithelium in response to the chronic irritation of molybdenum trioxide exposure. Pneumoconiosis has been described by Friberg (1979) in experimental animals exposed to molybdenum trioxide subchronically. Increased molybdenum intake by experimental animals has been shown to increase tissue levels of xanthine oxidase (Luo et al., 1983) which oxidizes hypoxanthine to xanthine and xanthine to uric acid in purine catabolism. During the process, superoxide anion radicals are generated and may lead to tissue damage. The present studies did not examine xanthine oxidase activity and superoxide radicals in the lung. In conclusion, exposure of rats to MoO3 by inhalation resulted in increased incidences of alveolar/bronchiolar adenoma or carcinoma (males), chronic inflammation in lung, hyaline degeneration of nasal respiratory epithelium, hyaline degeneration of olfactory epithelium (females), and squamous metaplasia of the epiglottis. Exposure of mice to MoO3 by inhalation resulted in increased incidences of alveolar/bronchiolar adenoma or carcinoma, metaplasia of alveolar epithelium, histiocytic infiltrate in lung (males), hyaline degeneration of the

MOLYBDENUM TRIOXIDE CARCINOGENESIS

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