Acrylonitrile Is a Multisite Carcinogen in Male and Female B6C3F1 Mice

Burhan I. Ghanayem,1, Abraham Nyska, Joseph K. Haseman and John R. Bucher

Environmental Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received December 26, 2001; accepted March 25, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acrylonitrile is a heavily produced unsaturated nitrile, which is used in the production of synthetic fibers, plastics, resins, and rubber. Acrylonitrile is a multisite carcinogen in rats after exposure via gavage, drinking water, or inhalation. No carcinogenicity studies of acrylonitrile in a second animal species were available. The current studies were designed to assess the carcinogenicity of acrylonitrile in B6C3F1 mice of both sexes. Acrylonitrile was administered by gavage at 0, 2.5, 10, or 20 mg/kg/day, 5 days per week, for 2 years. Urinary thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine were measured as markers of exposure to acrylonitrile. In general, there were dose-related increases in urinary thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine concentrations in all dosed groups of mice and at all time points. Survival was significantly (p < 0.001) reduced in the top dose (20 mg/kg) group of male and female mice relative to controls. The incidence of forestomach papillomas and carcinomas was increased in mice of both sexes in association with an increase in forestomach epithelial hyperplasia. The incidence of Harderian gland adenomas and carcinomas was also markedly increased in the acrylonitrile-dosed groups. In female mice, the incidence of benign or malignant granulosa cell tumors (combined) in the ovary in the 10 mg/kg dose group was greater than that in the vehicle control group, but because of a lack of dose response, this was considered an equivocal finding. In addition, the incidences of atrophy and cysts in the ovary of the 10 and 20 mg/kg dose groups were significantly increased. The incidences of alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in female mice treated with acrylonitrile at 10 mg/kg/day for 2 years. This was also considered an equivocal result. In conclusion, these studies demonstrated that acrylonitrile causes multiple carcinogenic effects after gavage administration to male and female B6C3F1 mice for 2 years.

Key Words: acrylonitrile; B6C3F1 mice; carcinogenicity; Harderian gland; forestomach; lung; ovary.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acrylonitrile is a heavily produced unsaturated nitrile. The estimated worldwide production of acrylonitrile in 1988 was 3,200,000 tons (IARC, 1999Go). The major uses of acrylonitrile involve the production of acrylic and modacrylic fibers, elastomers, acrylonitrile-butadiene-styrene and styrene-acrylonitrile resins, nitrile rubbers, gas barrier resins, and chemical intermediates such as acrylamide (IARC, 1999Go). Human exposure to acrylonitrile occurs primarily during its synthesis and processing for the manufacturing of fibers, resins, polymers, fibers, rubbers, and plastics (IARC, 1999Go).

Acrylonitrile is rapidly absorbed and distributed to all major tissues in animals. Acrylonitrile is metabolized via 2 major pathways (Burka et al., 1994Go; Sumner et al., 1999Go). The first pathway entails the direct conjugation of parent acrylonitrile with reduced glutathione (GSH). Subsequent degradation of this metabolite leads to the formation and urinary excretion of N-acetyl-S (2-cyanoethyl) cysteine (NACE; Burka et al., 1994Go, Fennell et al., 1991Go; Sumner et al., 1999Go). The second pathway involves oxidative metabolism of acrylonitrile leading to the formation of the epoxide intermediate, 2-cyanoethylene oxide (CEO). Recent studies using knock out mice demonstrated that CYP2E1 is the only enzyme responsible for acrylonitrile epoxidation (Ghanayem et al., 2000Go; Sumner et al., 1999Go). Subsequent metabolism of CEO occurs via conjugation with GSH or via hydrolysis to yield cyanide and other metabolites.

The genetic toxicity of acrylonitrile was reviewed by IARC (1999). Collectively, these studies suggested that acrylonitrile induces gene mutations, chromosomal aberrations, sister chromatid exchange, cell transformation, and increased unscheduled DNA synthesis, with the general requirement of an exogenous metabolism activating system (IARC, 1999Go; Leonard et al., 1999Go). Acrylonitrile is mutagenic in several strains of Salmonella typhimurium in the presence of an S9 metabolic activating system (Zeiger et al., 1987Go). In Chinese hamster ovary cells, acrylonitrile induced chromosomal aberrations in the presence or absence of exogenous metabolic activation (Danford, 1985Go; Gulati et al., 1985Go) and in Chinese hamster lung cells in the absence of a metabolic activating system (Ishidate and Sofuni, 1985Go).

Reported studies on the potential for the covalent binding of acrylonitrile with macromolecules are inconsistent. Acrylonitrile was shown to covalently bind with rat liver microsomal proteins in a time-dependent manner without metabolic activation (Peter and Bolt, 1981Go). Protein binding but not DNA binding of acrylonitrile apparently occurs via direct alkylation since it does not require NADPH (Guengerich et al., 1981Go). However, in the presence of NADPH, DNA binding was detectable (Guengerich et al., 1981Go). CEO also covalently binds to calf thymus DNA and rat microsomal proteins (Guengerich et al., 1981Go). Intraperitoneal administration of CEO resulted in binding to liver and brain proteins but not to DNA (Hogy and Guengerich, 1986Go). In another study, acrylonitrile failed to induce unscheduled DNA synthesis in rat liver and spermatocytes (Butterworth et al., 1992Go).

Early occupational epidemiology studies suggested some evidence of an association between exposure to acrylonitrile and human cancer. Generally, these early studies were inconclusive and more comprehensive epidemiology studies in occupationally exposed workers suggested that there is no significant association between exposure to acrylonitrile and carcinogenicity (Benn and Osborne, 1998Go; Swaen et al., 1998Go; Wood et al., 1998Go). In another recent study (Blair et al., 1998Go), mortality from lung cancer was slightly elevated in the group of workers with the highest cumulative exposure to acrylonitrile. However, further analysis of the exposure-response relationships did not support an association between exposure to acrylonitrile and lung cancer in humans.

In rats, a number of carcinogenicity studies using gavage, drinking water, or inhalation exposure were reported and reviewed (ATSDR, 1990Go; IARC, 1999Go; U.S. EPA, 1983Go). Collectively, these studies demonstrated that acrylonitrile is a multisite carcinogen; the target organs, which overlapped between these studies included the brain, spinal cord, forestomach, small intestine, tongue, mammary gland, and Zymbal's gland. Administration of 0, 20, 100, or 500 ppm acrylonitrile in drinking water to male Sprague-Dawley rats for 2 years resulted in a significant increase in the incidence of Zymbal's gland neoplasms (Gallagher et al., 1988Go). In another drinking water study, 0, 100, or 500 ppm acrylonitrile administered to Fischer 344 rats resulted in increased incidences of brain and spinal cord neoplasms (Bigner et al., 1986Go). Inhalation (Maltoni et al., 1988Go) or gavage (Maltoni et al., 1977Go) exposure to acrylonitrile also caused increased incidences of neoplasms in Sprague-Dawley rats. Other studies of acrylonitrile carcinogenicity, which also demonstrated similar effects in rats, were previously reviewed (ATSDR, 1990Go; IARC, 1999Go; U.S. EPA, 1983Go). No information on acrylonitrile carcinogenicity in any other animal species was available. The current studies were therefore designed to assess the carcinogenicity of this chemical in mice.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Full details concerning the materials and methods used in the present studies have been published in National Toxicology Program (NTP) Technical Report # 506 (NTP, 2001Go).

Chemical.
Acrylonitrile was purchased from Aldrich Chemical Company (Milwaukee, WI). The identity and purity of the chemical was assessed using elemental analyses, Karl Fischer water analyses, and gas chromatography. The overall purity of acrylonitrile was determined to be greater than 99%.

Dosing solutions.
Dosing solutions were prepared every 4 weeks by mixing acrylonitrile with deionized water. The stability of the dosing solutions was determined using gas chromatography and was determined to be at least 35 days for dose formulations stored in sealed vials at temperatures up to 28°C.

Animals.
Male and female B6C3F1 mice were obtained from Taconic Laboratory Animals and Services (Germantown, NY). Males were individually housed and females were housed 5/cage. Polycarbonate cages (Lab Products, Inc. Maywood, NJ) with Sani-Chip bedding (P. J. Murphy Forest Products Corp., Montiville, NJ) were used and changed once (males) or twice (females) weekly and rotated every 2 weeks; racks were changed and rotated every 2 weeks. NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA) and tap water were available ad libitum. Animals were housed in a facility that was maintained at 72 ± 3°F, relative humidity 50 ± 15%, 12-h light-dark cycle, and air changes of 10/h.

Animals were held for 11–12 days prior to the start of the study and were approximately 6 weeks old when the chemical administration started. All animals were observed twice daily and body weights were recorded at the beginning of the study, approximately every 4 weeks, and at the end of the study.

Two-year study design.
Groups of 50 male and 50 female B6C3F1 mice were administered acrylonitrile in deionized water by gavage at doses of 0, 2.5, 10, or 20 mg/kg, 5 days per week, for 105 weeks.

A complete gross necropsy and microscopic examination was performed on all mice. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 µm, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined. Major tissues/organs examined microscopically included the adrenal glands, bone, bone marrow, brain, clitoral gland, esophagus, gallbladder, Harderian gland, heart with aorta, kidney, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), liver, lung, mammary gland, lymph nodes (mandibular and mesenteric), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus, Zymbal's gland, and all gross lesions.

Analysis of urinary metabolites as markers of exposure.
Five male and 5 female mice per group were randomly selected for urine collection at 2 weeks and 3, 12, and 18 months. Mice were individually placed in metabolism cages for urine collection, and urine was collected over ice during a 24-h period, after which the mice were returned to their regular cages. The volume of urine was recorded, and urine creatinine concentrations were determined using a Hitachi 911 (Boehringer Mannheim, Indianapolis, IN) and reagents supplied by the manufacturer. Urine samples were stored frozen at –70°C or less until they were shipped to another facility for metabolite quantitation. Urinary thiocyanate was measured using a colorimetric method described by Pettigrew and Fell (1972). Urinary N-acetyl-S-(2-cyanoethyl)-L-cysteine was measured using liquid chromatography/mass spectrometry. Urine samples were treated with acetic acid and acetonitrile to precipitate proteins and then injected onto a strong anion exchange column (Keystone Excil SAX, 4.0 x 20 mm; Keystone Scientific, Bellefonte, PA). The mobile phase was 13.14% (0.01% ornithine in water), 86.74% acetonitrile, 0.04% acetic acid, and 0.08% triethylamine, and the system was operated isocratically at 1 ml/min. The mass spectrometer (Perkin Elmer Sciex, Norwalk, CT) monitored the peak areas of daughter ions at m/z 86 from the m/z 215 fragment of N-acetyl-S-(2-cyanoethyl)-L-cysteine relative to the m/z 100 daughter ion of the m/z 229 fragment of the internal standard (N-acetyl-S-(2-cyanoethyl)-L-cysteine) for 0.2 s each. The method was validated for concentrations of 1 µg/ml and above with acceptable precision, accuracy, and recovery.

Statistical analyses.
Analyses for possible dose-related effects on survival used Cox's (1972) method for testing 2 groups for equality and Tarone's (1975) life table test to identify dose-related trends. All reported values for survival analyses are 2 sided.

For the analyses of neoplastic and nonneoplastic lesion incidences, the Poly-3 test (Bailer and Portier, 1988Go; Piegorsch and Bailer, 1997Go; Portier and Bailer, 1989Go) was used. This test modifies the denominator of the tumor incidence rate to approximate more closely the total number of animal years at risk. Each animal is assigned a risk weight of 1 if it had a tumor or if it survived until the final sacrifice. Otherwise, its risk weight is the fraction of the entire study time it survived, raised to the third power. The summation of the individual animal risk weights is taken as the denominator in the poly-3 survival-adjusted rates reported in Tables 1–4GoGoGoGo.


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TABLE 1 Neoplastic and Nonneoplastic Lesions of the Forestomach in Mice Treated with Acrylonitrile for 2 Years
 

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TABLE 2 Neoplastic and Nonneoplastic Lesions of the Harderian Gland in Mice Treated with Acrylonitrile for 2 Years
 

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TABLE 3 Neoplastic and Nonneoplastic Lesions of the Ovary in Female Mice Treated with Acrylonitrile for 2 Years
 

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TABLE 4 Neoplastic and Nonneoplastic Lesions of the Lung in Female Mice Treated with Acrylonitrile for 2 Years
 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Survival and Body Weights
The effects of acrylonitrile on body weight and survival of B6C3F1 mice are shown in Figures 1 and 2GoGo. Mean body weights of 20 mg/kg males and females were generally less than those of the vehicle controls throughout most of the study. However, while the mean body weights of 20 mg/kg females were similar to the vehicle controls during the last 25 weeks of treatment, body weights of male mice declined and were 93% of controls at the end of dosing (Fig. 1Go). Survival of 20 mg/kg mice of both sexes was significantly (p < 0.01) less than that of the vehicle control groups (Fig. 2Go). Two-year survival rates were 38/50, 42/50, 39/50, and 14/50 for control, low-, mid-, and high-dose male mice, respectively. The corresponding survival rates for females were 39/50, 32/50, 39/50, and 23/50. There were no treatment-related signs of toxicity observed.



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FIG. 1. Effect of acrylonitrile on body weight gain of male and female B6C3F1 mice.

 


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FIG. 2. Effect of acrylonitrile on the survival of male and female B6C3F1 mice.

 
Urinary Metabolite Analyses
Urinary thiocyanate and NACE were measured as markers of exposure to acrylonitrile and as representatives of metabolites originating from acrylonitrile oxidation or direct conjugation of parent acrylonitrile to glutathione, respectively. In general, there were dose-related increases in urinary thiocyanate and NACE concentrations in all dosed groups of mice at 2 weeks and at 3, 12, and 18 months (Figs. 3 and 4GoGo). However, there was an increase in the excretion of each of the 2 metabolites at 12 and 18 months compared to 2 weeks and 3 months. Additionally, thiocyanate excretion was generally higher in the urine of female versus male mice (Fig. 3Go).



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FIG. 3. Urinary excretion of thiocyanate by male and female mice treated with acrylonitrile.

 


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FIG. 4. Urinary excretion of N-acetyl–S-(cyanoethyl)-L-cysteine by male and female mice treated with acrylonitrile.

 
Carcinogenicity
Forestomach.
The incidences of squamous cell papilloma, squamous cell carcinoma, and squamous cell papilloma or carcinoma (combined) of the forestomach occurred with positive trends in males and females, and the incidences in 10 and 20 mg/kg mice were significantly greater than those in the vehicle controls (Table 1Go). Squamous cell papillomas consisted of a pedunculated mass protruding into the lumen. Papillomas were composed of a stalk with numerous, branching, finger-like projections arising from the stalk (Fig. 5AGo). The projections were covered by one to many layers of squamous epithelium. Squamous cell carcinomas were well differentiated with some keratin pearl formation (Fig. 5BGo); however, some had evidence of submucosal invasion. Poorly differentiated squamous cell carcinomas were composed of spindle shaped cells without keratinization. Some squamous cell carcinomas metastasized primarily to the liver, but were also noted in the pancreas, spleen, kidney, lung, mesenteric lymph nodes, prostate gland, and adrenal gland.



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FIG. 5. (A) Squamous cell papilloma in the forestomach of a male mouse treated with 20 mg/kg/day of acrylonitrile. Note the presence of central stalk with secondary branches. (H&E, magnification x5). (B) Squamous cell carcinoma in the forestomach of a male mouse treated with 20 mg/kg/day of acrylonitrile. Note the presence of invasion of neoplastic cells into the submucosa. (H&E, magnification x5). (C) Focal squamous cell hyperplasia in the forestomach of a male mouse treated with 20 mg/kg/day of acrylonitrile. Note the presence of the thickened epithelium forming endophytic pegs. (H&E, magnification x10). (D) Adenoma in the Harderian gland of a male mouse treated with 20 mg/kg/day of acrylonitrile. Note the distinct nodule compressing the surrounding alveoli. (H&E, magnification x3.3). Dark arrow, adenoma; open arrow, normal Harderian gland tissue. (E) Marked hyperplasia in the Harderian gland of a male mouse treated with 20 mg/kg/day of acrylonitrile. Note the focus of tinctorially distinct cells (arrow), with no evidence of compression of the surrounding alveoli. (H&E, magnification x16). (F) Granulosa cell tumor in the ovary of a mouse treated with 20 mg/kg/day of acrylonitrile. The tumor cells, which have scanty cytoplasm, are forming varying sized pseudofollicular structures. The characteristic Call-Exner bodies (cell free area, see arrow) are also present. (H&E, magnification x10). (G) Severe atrophy in the ovary of a mouse treated with 20 mg/kg/day of acrylonitrile. Note the lack of follicles and corpora lutea and a predominance of interstitial tissue. (H&E, magnification x10). (H) Alveolar/bronchiolar carcinoma in a female mouse treated with 20 mg/kg/day of acrylonitrile. Note the localized invasiveness of the neoplastic cells (arrow). (H&E, magnification x25).

 
The incidences of mild focal or multifocal epithelial hyperplasia (combined) of the forestomach in 20 mg/kg males and females were significantly greater than those in the vehicle controls (Table 1Go); the incidence of mild diffuse or focal hyperkeratosis (combined) in 10 and 20 mg/kg males was significantly increased. Hyperplasia was characterized by thickened, orderly, and maturing epithelium. The thickened epithelium formed either endophytic pegs or closely apposed undulating folds (Fig. 5CGo). The hyperplastic lesions were often accompanied by focal, and occasionally diffuse, hyperkeratosis and were occasionally associated with chronic active inflammation. Hyperkeratosis without hyperplasia was rarely observed. Sporadic cases (1 or 2 per dose group) of multifocal ulceration of the forestomach were observed in 2.5 and 10 mg/kg males; the ulceration was generally accompanied by focal chronic active inflammation.

Harderian gland.
The incidences of Harderian gland adenoma and adenoma or carcinoma (combined) occurred with positive trends in males and females. The incidences in 2.5 mg/kg males and in 10 and 20 mg/kg males and females were significantly increased (Table 2Go). Loss of alveolar structure and minimal compression of the surrounding tissue characterized adenomas. These neoplasms were usually unilateral but were occasionally bilateral (Fig. 5DGo). Carcinomas generally resembled adenomas, but featured localized invasion of the structures adjacent to the gland and/or distant metastases. The incidences of Harderian gland hyperplasia in dosed groups of males and 10 and 20 mg/kg females were greater than those in the vehicle controls, and the incidence in 10 mg/kg males was significantly increased (Table 2Go). Hyperplasia was focal, without compression of the surrounding alveoli, and the cells tended to be tinctorially distinct and often larger than the normal surrounding cells. Due to the increased number of cells, the acinar walls appeared folded (Fig. 5EGo).

Ovary.
The incidence of benign or malignant granulosa cell tumors (combined) in the ovary of 10 mg/kg females was marginally (p = 0.06) greater than that in the vehicle control group (Table 3Go). The incidence in this group exceeded the historical range in NTP controls (all routes) given NTP-2000 diet (0–3%), and in water gavage or in feed controls given NIH-07 diet (0–2%). Further, the survival-adjusted rate was 0, 0, 8.5, and 2.8% in control, 2.5, 10, and 20 mg/kg treated mice, respectively (Table 3Go). One malignant granulosa cell tumor invaded the surrounding fat. Granulosa cell tumors are sex-cord stromal tumors derived from neoplastic transformation of the mesodermal follicular stem cells in the adult ovary. Benign granulosa cell tumors comprised the presence of varying sized follicles that compressed adjacent tissue or completely effaced the ovary (Fig. 5FGo). The follicles were composed of round to cuboidal cells arranged on a delicate basement membrane, and the cells often resembled granulosa cells of normal follicles. Granulosa cell tumors are the most common chemically induced ovarian neoplasms in NTP 2-year carcinogenicity studies.

The incidences of atrophy and cyst in the ovary of 2.5 (cyst only), 10, and 20 mg/kg females were significantly increased (Table 3Go); in dosed females, the severity of atrophy was marked, and the severity of cyst was mild. Atrophy was characterized by the partial to complete lack of histologically evident follicular and corpus luteum development and a predominance of interstitial tissue (Fig. 5GGo). Cysts were of follicular, epithelial inclusion, rete, or paraovarian types.

Lung.
The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) in 10 mg/kg females was significantly greater than that in the vehicle controls (Table 4Go). The incidences of alveolar/bronchiolar carcinoma in dosed groups and the incidences of alveolar/bronchiolar adenoma or carcinoma (combined) in 10 and 20 mg/kg females generally exceeded the historical ranges for controls (all routes) given NTP-2000 diet (0–12%) and for water gavage or in feed controls given NIH-07 diet (2–12%). The lower incidences of primary alveolar/bronchiolar adenomas and carcinomas in 20 mg/kg females may have been related to reduced survival in this group. The adenomas were distinct and compressing nodules that distorted the underlying alveolar structure, and the epithelial arrangement was irregular, papillary, glandular, or had solid patterns of cuboidal to columnar epithelium. In contrast to the adenomas, the alveolar/bronchiolar carcinomas (Fig. 5HGo) showed heterogenic growth patterns, cellular anaplasia with pleomorphism, nuclear atypia, localized invasiveness, or intrapulmonary or distant metastases. Incidences of alveolar epithelial hyperplasia were similar in vehicle control and dosed groups of females (Table 4Go). The incidences of alveolar/bronchiolar neoplasms in male mice were similar to those in the vehicle controls (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acrylonitrile was selected for toxicity and carcinogenicity testing in mice because of its extensive production and use, its high potential for human exposure, its classification as a probable human carcinogen, its carcinogenicity at multiple sites in rats, and the lack of carcinogenicity studies in a second animal species. Doses for this study were selected on the basis of the results of a 14-week toxicity study (NTP, 2001Go). Due to the high mortality in the 40 and 60 mg/kg groups and increased incidences of forestomach lesions in 40 mg/kg females (NTP, 2001Go), doses selected for the 2-year study in mice were 0, 2.5, 10, or 20 mg/kg per day. Because acrylonitrile was carcinogenic in rats regardless of the route of administration (gavage, drinking water, or inhalation) and because there were similarities in the target organs in these studies (ATSDR, 1990Go; IARC, 1999Go; U.S. EPA, 1983Go), gavage administration was selected as the exposure route in the current 2-year study.

Survival was significantly (p < 0.001) reduced in the top dose (20 mg/kg) group of male and female mice relative to controls (Fig. 2Go). The cause of deaths in the top dosed groups was thought to be due to squamous cell carcinoma of the forestomach, since 17/20 of these neoplasms occurred in animals dying prior to the end of the study. However, significant (p < 0.01) differences in survival remained even after adjusting for the early occurrence of these neoplasms. Importantly, the first forestomach squamous cell carcinoma was not observed until Day 513, but by that time there had already been 14 deaths in top dose males and 11 in top dose females compared with only a single death in the male and female control groups. Most of these early-dying animals did not have tumors, and the few neoplasms that were observed were unlikely to have been the cause of death. Thus, the major factor responsible for the early deaths appears to be the toxicity of acrylonitrile, with lethal forestomach squamous cell carcinoma likely being a contributing factor for those animals dying later in the study.

Urinary thiocyanate (marker of cytochrome p450 mediated metabolism) and N-acetyl-S-(2-cyanoethyl) cysteine (NACE; marker of direct acrylonitrile conjugation with acrylonitrile) were measured at various time points during the study. Excretion of each metabolite increased at 12 and 18 months compared to 2 weeks and 3 months. The reasons for these differences are unclear. However, these data confirmed that exposure of mice to acrylonitrile in the current study was accomplished as intended. Earlier reports suggested that acrylonitrile epoxidation is saturable (Kedderis et al., 1993Go); however, no evidence of saturation was detectable from the current results.

The incidences of forestomach squamous cell papilloma or carcinoma (combined) occurred with positive trends in male and female mice, and the incidences of these neoplasms in 10 and 20 mg/kg mice were significantly increased. Dose-related increases in the incidences of epithelial hyperplasia were also observed and were statistically significant in 20 mg/kg males and females. Forestomach tumors were observed in rats that received acrylonitrile by gavage, inhalation, or in drinking water (IARC, 1999Go). An association between chemical-induced forestomach epithelial hyperplasia and carcinogenesis has been proposed (Ghanayem et al., 1986Go) and proliferative epithelial forestomach lesions may constitute a continuum progressing to papilloma and then to carcinoma (Ghanayem et al., 1994Go). However, while increased incidences of epithelial hyperplasia of the forestomach occurred in mice that received 20 mg/kg acrylonitrile in the 2-year study, no such lesions were observed at lower doses that caused forestomach neoplasms. Further, forestomach hyperplasia was not observed in male or female mice at 20 mg acrylonitrile/kg or less in the 14-week study (NTP, 2001Go). Therefore, it is possible that this mechanism may not be the only contributing factor in the pathogenesis of forestomach carcinogenesis in mice. Acrylonitrile effects on cell proliferation and programmed cell death were investigated in the forestomach (target of carcinogenicity), glandular stomach, and liver of male F344 rats dosed with acrylonitrile by gavage for 6 weeks (Ghanayem et al., 1997Go). Acrylonitrile induced a net increase in mucosal cell proliferation in association with increased forestomach thickness. In contrast, acrylonitrile had no significant effect on cell proliferation in the liver or glandular stomach of treated rats. It was suggested that disruption of the balance between cell proliferation and programmed cell death in favor of a net enhancement of epithelial cell proliferation in the forestomach might have contributed to acrylonitrile carcinogenicity at this site.

Acrylonitrile also caused increases in the incidences of Harderian gland neoplasms in male and female mice in the 2-year study. Harderian gland hyperplasia also occurred in dosed males and females. Botts et al. (1999) suggested that primary hyperplasia is a precursor to the development of neoplastic lesions and it may be a contributing factor in chemical-induced Harderian gland carcinogenesis. In contrast to the present effects in mice, no increases in the incidences of Harderian gland tumors were reported in rats treated with acrylonitrile (IARC, 1999Go). However, Harderian gland tumors are uncommon in rats, suggesting that mice are more prone to chemical-induced tumors at this site.

Neoplasms of the ovary were also observed in female mice treated with acrylonitrile for 2 years. The incidence of benign or malignant granulosa cell tumor (combined) was slightly increased in 10 mg/kg females. The incidences of atrophy and cyst were significantly increased in 10 and 20 mg/kg females. In a recent review of over 400 NTP carcinogenicity studies, 8 chemicals were found to significantly increase ovarian neoplasms such as tubular adenomas, granulosa cell tumors, and mixed tumors in mice, but none have caused tumors in rats (Davis and Maronpot, 1996Go). Most of these chemicals are mutagenic, and most are associated with lung and/or mammary gland tumors. The relationship between ovarian and lung neoplasms is not clear. All 8 ovarian carcinogens also caused ovarian damage and epithelial hyperplasia, and these changes may precede development of ovarian neoplasia (Davis and Maronpot, 1996Go).

In the present study, acrylonitrile may also have caused increases in the incidences of lung neoplasms in female mice. The incidences of alveolar/bronchiolar adenoma or carcinoma (combined) in 10 and 20 mg/kg females were greater than those in the vehicle control group; the incidence in the 10 mg/kg group was significantly increased. There was a slight increase in the incidence of hyperplasia of the alveolar epithelium in dosed females. The low incidence of alveolar/bronchiolar adenoma or carcinoma (combined) in 20 mg/kg females may have been due to the decreased survival in these groups. No increases in the incidences of lung neoplasms in dosed male mice were observed. In contrast to the present effect in female mice, no increases in the incidences of lung neoplasms in acrylonitrile-treated rats have been observed in previous studies (IARC, 1999Go).

The carcinogenicity of acrylonitrile has been investigated extensively in gavage, drinking water, and inhalation studies in rats. Generally, acrylonitrile caused an increase in the incidences of brain, Zymbal's gland, forestomach, and mammary gland tumors. In the current 2-year studies, acrylonitrile caused increases in the incidences of forestomach and Harderian gland neoplasms in male and female mice and may have caused increases in the incidences of neoplasms of the ovary and lung in female mice. Therefore, it is clear that there are species differences in the target organs of acrylonitrile carcinogenicity. The forestomach is the only common target of acrylonitrile carcinogenicity in both species. In comparison to earlier studies in rats, there is no evidence of acrylonitrile carcinogenicity in the central nervous system in mice. It is well established that spontaneous brain tumors are rare in mice, and mice are apparently resistant to chemical-induced central nervous system tumors (Radovsky and Mahler, 1999Go).

The pattern of acrylonitrile-induced carcinogenicity in rats and mice resembles that observed in animals treated with 1,3-butadiene, vinyl chloride, benzene, or ethylene oxide (Melnick and Huff, 1993Go). While brain neoplasms were observed in rats treated with 1,3-butadiene, ethylene oxide, vinyl chloride, or acrylonitrile, lung neoplasms were reported only in mice. Zymbal's gland neoplasms were reported in rats treated with 1,3-butadiene, vinyl chloride, or acrylonitrile and in rats and mice treated with benzene. Mice appeared more sensitive to Harderian gland neoplasms and these neoplasms were observed in mice treated with 1,3-butadiene, benzene, or ethylene oxide. Ovarian neoplasms were also more prevalent in mice treated with 1,3-butadiene or benzene. Forestomach neoplasms were observed in mice treated with 1,3-butadiene or benzene and in rats treated with benzene or vinyl chloride (Huff et al., 1989Go; Melnick and Huff, 1993Go). All 5 chemicals are epoxides in nature (ethylene oxide) or are metabolized to mutagenic epoxide intermediates via cytochrome P450s (Ghanayem et al., 2000Go). Whether this property is related to the carcinogenic activity of these chemicals remains to be established.

Acrylonitrile CYP2E1-mediated metabolism was implicated in the mutagenicity and carcinogenicity of this chemical, and CEO is considered the ultimate carcinogenic metabolite. Significant species-dependent variations in the metabolism and disposition of acrylonitrile have been reported. The ratio of acrylonitrile epoxidation to direct GSH conjugation is greater in mice than in rats (Kedderis et al., 1993Go). Mice also excrete a significantly greater percentage of the administered dose as metabolites originating from CEO (Fennell et al., 1991Go), and excrete more urinary thiocyanate (Gut et al., 1975Go) than rats. Mouse hepatic microsomes metabolize acrylonitrile to CEO at higher rates than those of rats and humans, both of which epoxidize acrylonitrile at approximately the same rate (Roberts et al., 1991Go). It remains unclear how species differences in the metabolism of acrylonitrile may explain the differences in the target organs of acrylonitrile carcinogenicity in rats and mice.

The mechanism(s) of acrylonitrile-induced carcinogenicity remains uncharacterized. As described earlier, results of studies that described the binding of acrylonitrile with macromolecules were generally inconsistent. Acrylonitrile increased oxidative DNA damage in rat brain that was evident by the presence of 8-hydroxy-2`-deoxyguanosine, products of lipid peroxidation, and reactive oxygen species (Jiang et al., 1998Go). These effects were not observed in the liver of these animals. Additionally, in vitro incubation of high concentrations of acrylonitrile with a rat glial cell line or hepatocytes showed that 8-hydroxy-2`-deoxyguanosine levels and hydroxyl radical formation increased in rat glial cells but not in rat hepatocytes (Kamendulis et al., 1999aGo). In a recent study, acrylonitrile caused a dose-dependent inhibition of gap junctional intercellular communication in a rat astrocyte cell line (Kamendulis et al., 1999bGo), a property that is considered more characteristic of nongenotoxic carcinogens.

In conclusion, under the conditions of this study, there was clear evidence of carcinogenic activity of acrylonitrile in male and female B6C3F1 mice based on increased incidences of forestomach and Harderian gland neoplasms. Neoplasms of the ovary and lung were observed in female mice treated with acrylonitrile and may have been related to the administration of this chemical.


    ACKNOWLEDGMENTS
 
This study was conducted at Battelle Laboratories, Columbus Division, under an NIEHS contract.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (919) 541-4632. E-mail: ghanayem{at}niehs.nih.gov. Back


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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