Effects of the carcinogen, acrylonitrile, on forestomach cell proliferation ...

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Carcinogenesis vol.18 no.4 pp.675–680, 1997

Effects of the carcinogen, acrylonitrile, on forestomach cell proliferation and apoptosis in the rat: comparison with methacrylonitrile

B.I.Ghanayem1,3, M.R.Elwell and S.R.Eldridge2 1NIH/NIEHS, 3To

RTP, NC 27709, and 2PAI, Durham, NC, USA

whom correspondence should be addressed

Acrylonitrile (AN) and methacrylonitrile (MAN) are two major industrial nitriles used in the production of plastics and acrylic fibers. Whereas AN is a potent acute toxin and carcinogenic in rats, little is known regarding MAN. Current work is part of an overall effort designed to assess the potential toxicity/carcinogenicity of MAN. The present study compares the ability of the two chemicals to induce epithelial proliferation and apoptosis in the forestomach (FS; a target of AN carcinogenicity), liver and glandular stomach (non-targets of AN carcinogenicity) of male F344 rats. AN was administered to rats daily, by gavage, for 6 weeks, at 0.43 and 0.22 mmol/kg. MAN was administered at 0.87 and 0.43 mmol/kg. Both AN and MAN induced a dose-dependent increase in epithelial cell proliferation in the FS of male F344 rats as determined by bromodeoxyuridine (BrdU) incorporation into DNA. In contrast, AN, but not MAN caused a dose-dependent increase in the thickness of the forestomach squamous mucosa. This increased thickness (hyperplasia) was reflected by an increase in the number of total epithelial cells per unit length of mucosa. At doses of AN and MAN which induced a 2.3-fold increase in BrdU incorporation, apoptosis was 5- and 18-fold greater than controls, respectively. Although both MAN and AN caused a similar increase in cell proliferation, the relatively more prominent increase in the apoptotic index of the squamous epithelium of rats exposed to MAN may explain the lack of a detectable increase in the thickness of the mucosa compared to that seen with AN. The disruption of the balance between FS mucosal cell proliferation and apoptosis in favor of a net increase in the number of FS epithelial cells per unit length may contribute to the carcinogenicity of AN. In conclusion, present work demonstrated that AN selectively induced a net enhancement in FS cell proliferation, a site of its carcinogenicity. On the other hand, MAN-induced FS cell proliferation was associated with a parallel increase in apoptosis. The relatively greater increase in apoptosis by MAN may have compensated for the increase in FS mucosal cell proliferation and the lack of observable change in the FS thickness. Introduction Aliphatic nitriles (organic cyanides) are produced in large quantities for uses which include the production of plastics, elastomers, coatings and specialty plastics intended for use as food containers, syringes and medical/dental applications. The *Abbreviations: AN, acrylonitrile; CEO, 2-cyanothylene oxide; MAN, methacrylonitrile; UALI, unit area labeling index; LI, labeling index; ISEL, in situ end labeling. © Oxford University Press

high production and extensive use of aliphatic nitriles results in a significant potential for human exposure. The most used and investigated member of this family of chemicals is acrylonitrile (AN*). AN is a potent toxin and its effects in animals include acetylcholine-like toxicity, lung edema, liver and kidney damage, adrenal necrosis, gastric bleeding, and hematotoxicity (1–3). Chronic administration of AN by gavage (0.1–10 mg), inhalation (5–40 ppm) or in the drinking water (35–300 ppm) resulted in increased incidence of tumors in the brain, Zymbals gland, forestomach, tongue and mammary gland of rats (1–5). No studies on the carcinogenicity of AN were reported in other animal species. Epidemiological studies suggest that AN may be a human carcinogen (1–3,6). AN metabolism is known to proceed via the cytochrome P450 enzymes resulting in the formation of an epoxide intermediate (2-cyanoethylene oxide; CEO) (7,8). Both the parent molecule and CEO react with tissue glutathione in vivo (9,10), and with DNA in vitro. However, CEO was found to be more reactive with DNA (11,12). Furthermore, CEO and parent AN were shown to be mutagenic (13–15). The high reactivity of AN and CEO with DNA and glutathione are thought to contribute to the toxicity and carcinogenicity of AN (12,16). H2C5C-CN | H Acrylonitrile (AN)

H2C5C-CN | CH3 Methacrylonitrile (MAN)

Methacrylonitrile (MAN) is a structurally similar aliphatic nitrile which is produced in large quantities and it is used as a replacement for AN. The acute toxicity of MAN has some resemblance to that of AN and is apparently less potent (17). However, the carcinogenic potential of MAN remains unknown. MAN is thought to undergo metabolism via pathways qualitatively similar to that of AN (18–20). Recent work in our laboratory demonstrated that MAN is directly conjugated with glutathione or oxidized by cytochrome P450 enzymes to an epoxide intermediate (20). In addition, MAN depletes glutathione in vivo and in vitro (21). Mutagenicity studies suggested that MAN is not mutagenic in Salmonella or Drosophila in vitro (22,23). Since metabolic activation of AN is thought to play a role in its carcinogenicity and MAN is metabolized via similar pathways, present work was designed to characterize the effects of AN on cell proliferation and apoptosis in target (forestomach) and non-target organs (glandular stomach and liver) of its carcinogenicity and compare these effects with those induced by MAN in male rats. Materials and methods Chemicals Methacrylonitrile (MAN containing 1% hydroquinone as a stabilizer) and acrylonitrile (AN containing 1% hydroquinone as a stabilizer) were purchased

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B.I.Ghanayem, M.R.Elwell and S.R.Eldridge from Fluka (Ronkokoma, NY). Dosing solutions were prepared in tap water in a dose volume (gavage) of 5 ml/kg/day, and immediately used to prevent possible polymerization. Animals and treatments Three-month-old male Fischer 344 rats (Charles River Breeding Laboratories, Raleigh, NC) were used throughout the present studies. Animals were quarantined for a week before use in temperature- and humidity-controlled rooms with a 12 h light:dark cycle. NIH diet #31 and water were available ad libitum throughout the experiments. All animal care and experimentation were conducted according to the NIH guidelines (24). Animals were treated in groups as follows: Group Group Group Group Group

1 2 3 4 5

(MH): 12 rats received 0.87 mmol MAN/kg/day for 6 weeks. (ML): 12 rats received 0.43 mmol MAN/kg/day for 6 weeks. (AH): 12 rats received 0.43 mmol AN/kg/day for 6 weeks. (AL): 12 rats received 0.22 mmol AN/kg/day for 6 weeks. (C): 12 rats received 5 ml water/kg/day for 6 weeks.

Eighteen hours prior to death, six rats from each treatment group were implanted subcutaneously with osmotic minipumps containing bromodeoxyuridine (BrdU). Tissues from this group of animals was used to assess BrdU incorporation in the stomach. The remaining six rats from each dose group were implanted with osmotic minipumps 5 days before death (during which chemical administration continued). Tissues from these animals were use to assess BrdU incorporation in hepatocytes. Alzet Osmotic minipumps (model 2002 Alza Corporation, Palo Alto, CA) were filled with a solution of 30 mg

BrdU/ml distilled water and primed for 2–3 h in sterile saline before implantation using sterile techniques under methoxyflurane (metofane; PitmanMoore, Inc., Washington Crossing, NJ) anesthesia by inhalation. At the time of death, animals were euthanized under CO2. Stomachs were dissected free of other tissues, opened along the lesser curvature, pinned flat to a piece of cardboard, washed and immediately fixed in 70% cold ethanol. A middle radial section from the right anterior lobe of the liver, and a 1-cm length section of the small intestine were also removed and fixed in 70% cold ethanol. The glandular and forestomachs were trimmed and processed for preparation of histologic sections. Histopathological evaluation of forestomach Based upon microscopic examination of H&E stained sections of forestomachs, a minimal to mild hyperplasia (thickening) of the squamous mucosa was evident in the AN exposed animals (data not shown). Forestomach squamous epithelial proliferation was evaluated with light microscopy by determining the number of cells (nuclei) per unit length muscularis and by quantitating BrdU-stained cells. The total number of epithelial cells per unit length of muscularis mucosa was quantitated in four 0.25-mm randomly selected fields and used as an index of forestomach proliferation. No histopathological lesions were observed in the glandular stomach or the liver of control or chemical treated animals. Immunohistochemical evaluation of cell proliferation Serial sections of 5 µm were cut and mounted onto poly-1-lysine coated slides. A section of duodenum was included on each slide as a positive control for BrdU staining. Mounted tissues were deparaffinized and dehydrated, and

Fig. 1. Effects of AN and MAN on the body weight of male F344 rats. AN was administered by gavage at 0.22 and 0.43 mmol/kg/day for 6 weeks. MAN was similarly administered at 0.43 and 0.87 mmol/kg/day. Control rats received water at 5 ml/kg/day. Values are presented as the mean 6 SE of 12 animals. Asterisk indicates values that are statistically significant from controls.

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Acrylonitrile as a carcinogen ISEL staining. Cells that were positively stained by the ISEL technique were identified by brown to black pigmented nuclei. The number of apoptotic cells as determined by positive ISEL staining were counted in 16 randomly selected 0.25-mm fields of forestomach mucosa and the apoptotic index is expressed as the number of apoptotic cells per unit length of the muscularis mucosa. Statistical analysis Statistical comparisons of chemical-treated versus vehicle-treated groups were performed using a pairwise analysis of variance (pooled t-test). Values were considered statistically significant at P ø 0.05.

Results

Fig. 2. H&E stained sections of the forestomach of male rats which received water vehicle at (A) 5 ml/kg/day; (B) 0.43 mmol AN/kg/day; or (C) 0.87 mmol MAN/kg/day by gavage. Epithelial hyperplasia observed in acrylonitrile but not methacrylonitrile treated rats. (3200.) stained immunohistochemically for the identification of cells with BrdU incorporation during S-phase as previously described (25). BrdU incorporation was quantitated in the forestomach, glandular stomach and liver using light microscopy. In the forestomach, the unit length labeling index (ULLI) was presented as the number of BrdU-labeled epithelial cells per mm muscularis mucosa and scored in four 0.25 mm randomly selected fields. In the glandular stomach, the unit area labeling index (UALI) was assessed as the number of BrdU-labeled epithelial cells per square mm and scored in four 0.25 mm2 randomly selected areas. The labeling index (LI) in the liver was assessed as the percentage of BrdU-labeled hepatocytes scored in 10 fields; at least 2000 hepatocytes were counted per animal. In situ end labeling (ISEL) Tissue sections of 5 µm thickness were mounted on positively charged slides (Superfrost Plus, Fischer Scientific, Pittsburgh, PA). ISEL of DNA was performed with minor modifications of previously described methods (26). A section of duodenum was included on each slide as a positive control for

Gavage administration of AN or MAN to male F344 rats resulted in a dose-dependent reduction of body weight gain (Figure 1). This effect was evident at 2 weeks after chemical administration and persisted as long as chemical exposure continued. At the low doses (0.22 mmol AN/kg and 0.43 mmol MAN/kg), the two chemicals caused minimal decrease in body weight gain. In contrast, daily administration of the high doses (0.43 mmol AN/kg and 0.87 mmol MAN/kg) resulted in a more pronounced and significant depression of weight gain throughout the 6-week study. Comparison of the body weight gain of rats which received equimolar doses of AN and MAN (0.43 mmol/kg) for 6 weeks showed that AN had a more pronounced effect than MAN on body weight gain. Microscopic examination revealed a minimal to mild hyperplasia of the squamous mucosa of the forestomach of rats exposed to AN (Figure 2). In the low AN dose group, this diffuse change, generally of minimal severity, consisted of a slight, statistically insignificant, increase of forestomach thickness (7% above control; Figure 5). The superficial layers of keratin fibers on the mucosal surface were also slightly increased in thickness relative to controls. In the high AN dose group, forestomach hyperplasia was of mild severity with an increased thickness to 9–12 cell layers or greater compared to controls (Figure 2B). Forestomach hyperplasia (expressed as the increase in the total number of epithelial cells per mm muscularis) in the AN high dose group was ~60% above vehicle-treated controls (Figure 5). The surface keratin layer was also increased in this dose group relative to controls. No significant hyperplasia was observed in the forestomach of rats in the MAN groups (Figures 2 and 5). In addition, examination of H&E stained glandular stomach and liver sections from AN- or MAN-treated animals revealed no morphological changes. The effects of AN and MAN on cellular proliferation in the forestomach of male F344 rats were assessed by quantitative determination of BrdU incorporation into S-phase DNA using immunohistochemical staining. Both AN and MAN induced a dose-dependent increase in forestomach squamous mucosal cell proliferation (Figure 3). The increase in forestomach mucosal cell proliferation was significant at both the low and high doses of both chemicals (Figure 5). At equimolar doses (0.43 mmol/kg), AN induced a greater increase in epithelial cell proliferation than MAN. In comparison to tissues from vehicle-treated rats, no chemical-related increase in the incorporation of BrdU into S-phase DNA was detected in the liver or glandular stomach of AN or MAN-treated groups (data not shown). The effects of AN and MAN on apoptosis as determined by in situ end labeling of tissue sections is shown in Figures 4 and 5. As shown in Figure 4, apoptotic bodies were observed in the forestomach of AN and MAN treated rats only at the high doses of the two chemicals. In comparison to vehicle 677

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Fig. 3. BrdU immunostained sections of forestomachs of male rats which received water vehicle at (A) 5 ml/kg/day; (B) 0.43 mmol AN/kg/day; or (C) 0.87 mmol MAN/kg/day. Increased number of proliferating epithelial cells (dark nuclei) are present in acrylonitrile (AN) and methacrylonitrile (MAN) treated rats. (3200.)

Fig. 4. Comparison of the in situ end labeling (ISEL) of forestomach sections of male rats which received (A) water vehicle; (B) 0.43 mmol AN/ kg/day; or (C) 0.87 mmol MAN/kg/day. Increased apoptotic bodies (dark nuclei) are present in the forestomach of AN and MAN treated rats. (3200.)

treated controls, the forestomach apoptotic index was significantly higher in the high dose groups of both chemicals than in the vehicle-treated group (Figure 5). No increase in apoptosis was detectable in the liver or glandular stomach of vehicle or chemical treated rats (data not shown). Comparison of the relationship between forestomach hyperplasia and apoptosis revealed that while MAN induced no hyperplastic changes in the rat forestomach, a significant increase in the forestomach unit length apoptotic index was detected at the high MAN dose only (Figure 5). In contrast, AN induced a significant increase in forestomach apoptosis (albeit much smaller than that induced by the high dose of

MAN) at the high dose that was coupled with an increase in hyperplasia.

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Discussion Acrylonitrile (AN) is a multi-site carcinogenic aliphatic nitrile in rats. Chronic administration of this chemical by gavage, inhalation, or in the drinking water resulted in increased incidence of tumors in the brain, Zymbals gland, forestomach, tongue and mammary gland of rats (1–3, 27). MAN is a structurally similar aliphatic nitrile. Work conducted in this laboratory in recent years demonstrated great similarities between the metabolism of AN and MAN (19,20).

Acrylonitrile as a carcinogen

Fig. 5. The relationships between forestomach epithelial thickness (hyperplasia), epithelial cell proliferation, and apoptosis. Rats received acrylonitrile at 0.43 mmol/kg/day (AH) and 0.22 mmol/kg (AL), and methacrylonitrile at 0.87 mmol/kg/day (MH) and 0.43 mmol/kg (ML). Control animals (C) received vehicle at 5 ml/kg. Values are presented as the mean 6 SE of six animals. Asterisk indicates values that are statistically significant from controls.

Similar to AN, MAN is metabolized via an epoxide intermediate in a reaction presumably catalysed by the cytochrome P450 (19,20). There is evidence that metabolism of AN to CEO and the interaction with DNA may contribute to the toxicity and carcinogenicity of AN (11–13). Our work demonstrated that MAN is also metabolized via an epoxide intermediate. Since the carcinogenicity of MAN remains uncharacterized, we hypothesized that MAN may cause qualitatively similar toxicity/carcinogenicity as AN and MAN may target the same organs which were targeted by AN after chronic administration. Recent advances in understanding the mechanisms of chemically-induced cancer suggested that alterations in the normal balance between cell proliferation and apoptosis (genetically programmed cell death) by chemicals may play a role in the pathogenesis of tumors (28,29). Since AN is a known carcinogen in rats, the current investigations were designed to compare the effects of AN and MAN on cell proliferation and apoptosis in a target (forestomach) and non-target (liver and glandular

stomach) organs of AN carcinogenicity. Quantitative assessment of the effect of the two aliphatic nitriles on cell proliferation indicated that AN induces a dose-dependent increase in S-phase DNA synthesis (as evident by increased BrdU incorporation) in a target organ for its carcinogenicity, forestomach. This increase in cell proliferation was also associated with a dose-dependent increase in the thickness (hyperplasia) of the forestomach epithelial cell layer. No increase in cell proliferation was detected in non-target organs (liver and glandular stomach). Additionally, AN-induced cell proliferation was associated with a disproportionately smaller increase in apoptosis at the high dose only. A net increase in favor of forestomach cell proliferation apparently prevailed and was reflected by increased stomach thickness by AN. These data demonstrate for the first time that carcinogenic doses of AN selectively induced cell proliferation at a target of its carcinogenicity and suggested that increased epithelial cell proliferation may contribute to the development of forestomach tumors by this chemical. These effects of AN are in agreement with earlier conclusions that a net sustained increase in cell proliferation may play a role in chemical-induced forestomach carcinogenicity (30,31). Similar associations between cell proliferation and carcinogenicity were reported with other known forestomach carcinogens including diglycidyl resorcinol ether, 1-chloro-2methylpropene (dimethylvinyl chloride), 3-chloro-2-methylpropene and ethyl acrylate (31). In particular, we recently demonstrated that a net increase in cell proliferation was sustained at the target of ethyl acrylate carcinogenicity, forestomach (30). Ethyl acrylate had no effect on apoptosis in rat forestomach (our unpublished findings). Furthermore, no enhancement of cell proliferation was detected in non-target organs of ethyl acrylate such as the liver and glandular stomach (30). It was concluded that the selective sustained increase in forestomach epithelial cell proliferation played a role in the pathogenesis of forestomach tumors induced by this chemical. Present work also demonstrates that MAN increased forestomach S-phase DNA synthesis in a dose-dependent manner. However, MAN is apparently less efficacious than AN in rats. Similar to AN, no increase in cell proliferation was detected in the liver or glandular stomach of MAN-treated male rats (data not shown). In addition, while MAN increased cell proliferation at both doses, it resulted in a proportional increase in apoptosis at the high dose only. The overall effect of the low dose of MAN is similar to the response observed at the low dose of AN (both significantly increased forestomach cell proliferation with no effect on apoptosis or thickness). Considering the fact that the low AN dose is tumorigenic in the rat forestomach (1–3,27), it is possible that the low MAN dose, when administered to rats chronically by gavage, may cause forestomach tumors as well. The increase in forestomach cell proliferation by the high dose of MAN apparently triggered a compensatory increase in forestomach apoptosis resulting in some parity between the two responses. This suggestion is supported by the finding that, under the current experimental conditions, MAN caused no increase in the thickness of the forestomach mucosa. Since MAN resulted in an increase in cell proliferation accompanied by a compensatory increase in apoptosis at the high dose only, it is theoretically possible that, if similar balance between cell proliferation and apoptosis is sustained after chronic administration, this chemical may exhibit a greater carcinogenic 679

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potency at the low dose compared to the high dose. In fact, another forestomach carcinogen, dimethylvinyl chloride, exhibited a reverse dose–response relationship (significantly increased the incidence of forestomach tumors at the low dose (100 mg/kg/day) with no tumors observed at the high dose (200 mg/kg/day) in male rats (32). In conclusion, it is thought that perturbation of the normal balance between cell proliferation and apoptosis in favor of enhanced cell proliferation and hyperplasia plays a role in the induction of tumors by a number of chemicals. It is therefore speculated that AN-induced cell proliferation plays a role in the pathogenesis of tumors caused by this chemical. This suggestion is supported by the fact that these changes in the growth dynamics of the forestomach mucosa by AN occurred at doses known to produce forestomach tumors and were selective to a target of its carcinogenicity (forestomach). In comparison to AN, MAN resulted in a net enhancement of mucosal cell proliferation in the rat forestomach, however, no mucosal thickening was observed at this site. It is likely that induction of forestomach apoptosis by MAN provided a compensatory response for the increased mucosal forestomach proliferation which resulted in no observable thickening of the forestomach epithelium. Whether chronic administration of MAN will result in a similar change in the balance between cell proliferation and apoptosis in the forestomach and lead to tumor development remains to be established. Additional work to assess the kinetics of cell proliferation, apoptosis, and organ growth at a wider range of AN and MAN doses will be necessary before these growth related biological processes can be utilized to more accurately predict chemical-induced carcinogenicity. The utility of data obtained from this work will be enhanced by studies currently underway by the NTP to characterize the carcinogenicity of MAN. In the mean time, as more mechanism-based toxicology studies become available and include more chemicals, the use of this approach and the utility of such biological end points may prove beneficial in hazard identification and in cancer risk assessment. References 1. US Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry (1990) Toxicological Profile for Acrylonitrile. TP-90–1. USDHHS, New York. 2. US Environmental Protection Agency (1983) Health Assessment Document for Acrylonitrile EPA-600/8–82–007. USEPA, Washington DC. 3. World Health Organization (1983) Acrylonitrile: Environmental Health Criteria # 28. WHO, Geneva. 4. Bigner,D.D., Bigner,S.H., Burger,P.C., Shelburne,J.D. and Friedman,H.S. (1986) Primary brain tumors in Fischer 344 rats chronically exposed to acrylonitrile in their drinking-water. Fd Chem. Toxicol., 24, 129–137. 5. Maltoni,C., Ciliberti,A., Cotti,G. and Perino,G. (1988) Long term carcinogenicity bioassays on acrylonitrile administered by inhalation and by ingestion to Sprague–Dawley rats. Annl. NY Acad. Sci., 534, 179–202. 6. IARC Working Group (1987) Acrylonitrile: IARC Monograph. Eval. Carcinog. Risk. Chem. Human, Suppl. 7, 79–80. 7. Langvert,P.W., Putzig,C.L., Braun,W.H. and Young,J.D. (1980) Identification of the major urinary metabolites of acrylonitrile in the rat. J. Toxicol. Environ. Hlth, 6, 273–282. 8. Ghanayem,B.I. and Ahmed,A.E. (1982) In vivo biotransformation and biliary excretion of 1-14C-acrylonitrile in rats. Arch. Toxicol., 50, 175–185. 9. Hashimoto,K. and Kanai,R. (1972) Effect of acrylonitrile on sulfhydryls and pyruvate metabolism in tissues. Biochem. Pharmacol., 21, 635–640. 10. Dudley,H.C. and Neal,P.A. (1942) Toxicology of acrylonitrile: I. A study of acute toxicity. J. Ind. Hyg. 24, 27–36. 11. Hogy,L.L. and Guengerich,F.P. (1986) In vivo interaction of acrylonitrile and 2-cyanoethylene oxide with DNA in rats. Cancer Res., 46, 3932–3938. 12. Guengerich,F.P., Geiger,L.E., Hogy,L.L. and Wright,P.L. (1981) In vitro metabolism of acrylonitrile to 2-cyanoethylene oxide, reaction with

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