Effects of butylated hydroxyanisole, butylated hydroxytoluene, and NaCl on gastric carcinogenesis initiated with N-methyl-N'-nitro-N-nitrosoguanidine in F344 rats

Promoting activities of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and NaCl and of combinations of these antioxidants with NaCl on gastric carcinogenesis initiated by N-methyl-N'-nitro-N-nitrosoguanidine [(MNNG) (CAS: 70-25-7; 1-methyl-3-nitro-1-nitrosoguanidine] were investigated in male inbred F344 rats. Animals, 6-week old, were given an intragastric administration of MNNG at 150 mg/kg body weight by gastric tube and 1 week later were placed on a diet containing BHA (0.5%), BHT (1.0%), NaCl (5.0%), BHA (0.5%) plus NaCl (5%), or BHT (1.0%) plus NaCl (5.0%) for 51 weeks. Control rats received no further treatment after MNNG administration. A single intragastric application of MNNG to rats induced multiple epithelial tumors of the forestomach and a few epithelial tumors of the glandular stomach after 52 weeks. Squamous cell carcinomas of the forestomach were seen in 2 of 18 effective rats (11.1%) in the control groups, and the incidences in the groups receiving the subsequent treatment were 45.0% with BHA, 15.8% with BHT, 30% with NaCl, 70% with BHA plus NaCl, and 52.9% with BHT plus NaCl. Differences in the incidences of squamous cell carcinoma between the controls and groups given BHA, BHA plus NaCl, and BHT plus NaCl were statistically significant. NaCl given alone after MNNG administration also significantly increased the incidence of papillomas in the forestomach. Incidences of glandular stomach tumors, adenomas and carcinomas were not affected by any of the subsequent treatments. No tumors of the stomach developed in the groups given BHA, BHT, and NaCl without MNNG pretreatment. Thus the present experiment revealed that BHA and NaCl but not BHT exert promoting activity on MNNG-induced forestomach carcinogenesis in rats and that, when BHA and BHT were given with NaCl, promotion was more marked, suggesting a synergistic effect on tumor promotion.     

Modifying effects of concomitant treatment with butylated hydroxyanisole or butylated hydroxytoluene on N,N-dibutylnitrosamine-induced liver, forestomach and urinary bladder carcinogenesis in F344 male rats

The modifying effects of concomitant antioxidant treatment on N,N-dibutylnitrosamine (DBN)-induced carcinogenesis were investigated. Male F344 rats were given 0.05% DBN in their drinking water for 16 weeks, and simultaneously administered powder diet containing 2.0% butylated hydroxyanisole (BHA) or 0.7% butylated hydroxytoluene (BHT) for 16 weeks. Control animals received drinking water containing 0.05% DBN without antioxidant treatment. The final incidences of hepatocellular carcinomas were 100, 100 and 40% in the DBN plus BHA, DBN plus BHT and DBN treated groups, respectively, the difference being significant (P less than 0.001). Lung metastases were only observed in the DBN plus BHT group and DBN plus BHA group (50%, P less than 0.001; 7%, respectively). The incidence of papillary or nodular hyperplasia of the urinary bladder in the DBN plus BHA group was significantly higher than that of the control (P less than 0.05). Furthermore, esophageal carcinomas and papillomas were observed in all DBN treated groups, with no inter-group significant variation in yield. On the other hand, combination of DBN treatment with BHA or BHT significantly reduced the resultant incidences of forestomach hyperplasia. The results clearly demonstrated that concomitant administration of antioxidants, and in particular BHT, can modify DBN carcinogenesis.     

Dose-dependent effects of butylated hydroxyanisole, butylated hydroxytoluene and ethoxyquin for promotion of bladder carcinogenesis in N-butyl-N-(4-hydroxybutyl)nitrosamine-initiated, unilaterally ureter-ligated rats

Dose-dependent effects of 3 antioxidants, butylated hydroxyanisole (BHA, 2.0, 1.0 and 0.5%), butylated hydroxytoluene (BHT, 1, 0.5 and 0.25%) and ethoxyquin (0.5, 0.25 and 0.125%) on the development of preneoplastic lesions in the bladder of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-treated rats were investigated. Feeding of the antioxidants after pretreatment of 0.05% BBN commenced and unilateral ureteric ligation was combined at week 3 of the experiment. Surviving rats were killed at the end of week 24. BHA and BHT, but not ethoxyquin increased dose-dependently the incidence and number of preneoplastic lesions, papillary or nodular hyperplasia of the urinary bladder in rats treated with BBN. Particularly, the incidence and number of PN hyperplasia in rats treated with 2.0% BHA and 1.0% BHT were significantly higher than those of the control group. Thus, promoting activities of BHA and BHT, but not ethoxyquin for the urinary bladder were confirmed in this system of BBN-initiated, unilaterally ureter-ligated rats.     

Modifying effects of simultaneous treatment with butylated hydroxyanisole (BHA) on rat tumor induction by 3,2'-dimethyl-4-aminobiphenyl, 2,2'-dihydroxy-di-n-propylnitrosamine and N-methylnitrosourea

The modifying effects of concurrent treatment with high or low doses of butylated hydroxyanisole (BHA) on wide-spectrum carcinogen-induced carcinogenesis were studied in male F344 rats. Groups of 20 animals were treated with 2 or 0.04% BHA for 24 weeks. Starting 2 weeks after the commencement of BHA treatment, they were given s.c. injection of 50 mg/kg body weight 3,2'-dimethyl-4-aminobiphenyl (DMAB) once a week, i.g. administrations of 200 mg/kg body weight 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN) once every 2 weeks, or i.p. injection of 15 mg/kg body weight N-methylnitrosourea (MNU) once every 2 weeks for 22 weeks. Further groups of rats were treated with DMAB, DHPN, MNU, or 2 or 0.04% BHA alone. All surviving animals were killed 24 weeks after the beginning of the experiment and the target organs examined histopathologically. The BHA treatment dose-dependently decreased the incidence of DMAB-induced liver preneoplastic lesions but was associated with significant tumor induction in the forestomach (papillomas, 40%, P less than 0.01) and urinary bladder (papillomas, 53%, P less than 0.001; carcinomas, 80%, P less than 0.001), where no lesions were observed in the group given only DMAB. Concurrent administration of 2% BHA also significantly inhibited the development of alveolar hyperplasia (P less than 0.001) of the lung in DHPN-treated animals, while enhancing induction of forestomach papillomas (P less than 0.05) and simple hyperplasia in the urinary bladder. Neither MNU nor 2% BHA alone induced forestomach carcinoma or papillary or nodular hyperplasia (PN hyperplasia) in the urinary bladder. However, these lesions were observed in 100% (P less than 0.001) and 55% (P less than 0.001) of animals respectively, receiving the two compounds in combination. These results demonstrated that concurrent treatment with BHA not only inhibits but can also strongly enhance carcinogenesis depending on the organ, irrespective of whether the carcinogens act directly or require metabolism. The finding that BHA potently modified carcinogenesis at 0.04% in diet, 1/50 of the carcinogenic dose, suggests that actual dietary levels close to the human situation might play a significant role in tumor development in man.     

Modification by antioxidants and p, p'-diaminodiphenylmethane of 7, 12-dimethylbenz[a]anthracene-induced carcinogenesis of the mammary gland and ear duct in CD rats

The effects of antioxidants on mammary gland carcinogenesispretreated with 7, 12-dimethylbenz[a]anthracene (DMBA) in femaleSprague-Dawley rats were examined. The antioxidants used werebutylated hydroxyanisole (BHA), butylated hydr-oxytoluene (BHT),sodium L-ascorbate, -tocopherol, ethoxy-uin and p, p’-diaminodiphenylmethane(DDPM), which is an inhibitor of carcinogenesis in the liver,kidney and urinary bladder. Female Sprague-Dawley rats of 50days old were treated with 2.5 mg/100 g body weight of DMBA,and from 1 week later were given diet supplemented with 1% BHA,0.7% BHT, 5% sodium L-ascorbate, 1.5% -tocopherol, 0.5% ethoxyquinor 0.1% DDPM for 33 weeks and then killed. The incidences ofmammary tumors, carcinomas and fibroaden-omas in DMBA-treatedanimals were reduced by diet containing BHA or ethoxyquin. Dietcontaining BHT or DDPM inhibited the induction of only fibroadenomas.The incidence of ear duct tumors in DMBA-treated animals wasreduced by diet containing BHT, -tocopherol or ethoxyquin.     

Ornithine decarboxylase activity and DNA synthesis in rats after long term treatment with butylated hydroxyanisole, sodium saccharin or phenobarbital

Ornithine decarboxylase (ODC) activity and DNA synthesis were measured in the forestomach, urinary bladder and liver of rats given diet containing 2% butylated hydroxyanisole (BHA), 5% sodium saccharin (SS) or 0.05% phenobarbital (PB) for 21 weeks. ODC activity was not increased significantly in any of these organs in any of the groups, but the labeling index (LI) of the forestomach epithelium was increased by BHA and PB, and that of the urinary bladder epithelium by SS. PB did not increase the LI of the liver. These results do not indicate any relation between the effects of promoters, and the ODC activity or DNA synthesis in non-initiated target organs.     

Promoting activities of butylated hydroxyanisole and butylated hydroxytoluene on 2-stage urinary bladder carcinogenesis and inhibition of {gamma}-glutamyl transpeptidase-positive foci development in the liver of rats

Butylated hydroxyanisole (BHA) and butylated hydroxy-toluene(BHT) were evaluated for possible promoting activity for urinarybladder carcinogenesis in male F344 rats initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine(BBN). The rats were treated with 0.01 or 0.05% BBN in the drinkingwater for 4 weeks and then administered 2% BHA or 1% BHT inthe diet for 32 weeks. Surviving rats were killed at the endof week 36 of the experiment. The incidences of cancer and papillomaand the average number of cancers, papillomas and papillaryor nodular hyperplasias (PN hyperplasias) per 10 cm of basementmembrane were significantly increased in the group receivingBHA following initiation by 0.05% BBN compared with the groupgiven BBN only. BHT also significantly increased these lesionsof the bladder, but not the average number of cancers, in ratstreated with 0.05% BBN. The ability of four antioxidants, BHA,BHT, sodium L-ascorbate (ascorbate) and ethoxyquin, to promotethe induction of -glutamyllranspeplidase (-GT)-positive fociinitiated by diethylnitrosamine (DENA) in the liver of F344rats was tested. Rats were given a single i.p. injection of200 mg/kg body weight of DENA, and 2 weeks later the animalswere exposed to 2% BRA, 1% BHT, 5% ascorbate or 1% ethoxyquin,respectively, in the diet for 6 weeks. All animals were subjectedto partial hepatectomy at the end of week 3. The number of -GT-positivefoci in the groups fed either BHA, BBT or ethoxyquin after DENAwere significantly decreased compared with the control group.These findings show that BRA and BHT are promoters for the urinarybladder carcinogenesis initiated by BBN, but that these andother antioxidants significantly inhibit the induction of -GTpositive foci in the liver.     

Modulation of azaserine-induced pancreatic foci by phenolic antioxidants in rats

Effects of the dietary phenolic antioxidants butylated hydroxyanisole [(BHA) CAS: 25013-16-5; (1,1-dimethylethyl)-4-methoxyphenol] and butylated hydroxytoluene [(BHT) CAS: 128-37-0; 2,6-di-tert-butyl-p-cresol] on pancreatic tumorigenesis were examined. Male LEW inbred rats were given injections of 30 mg azaserine [CAS: 115-02-6; diazoacetate (ester) serine] per kg body weight once a week for 3 weeks and maintained on either a control diet or 0.45% BHA- or 0.45% BHT-supplemented control diet throughout the initiation and post-initiation phases of the experiment. At 4 months post initiation, pancreatic tissue sections were quantitatively examined for the number and size of preneoplastic foci. BHT and BHA treatments reduced the number of acidophilic foci per pancreas by 32 and 48%, respectively, but were without effect on focal size. By contrast, basophilic foci were not subject to modulation by these antioxidants. A constellation of enzyme activities involved in carcinogen inactivation and known to be perturbed by antioxidant treatment was examined in liver and pancreas. The hepatic activities of glucose-6-phosphate dehydrogenase, glutathione reductase, and glutathione-S-transferases were markedly elevated while catalase and superoxide dismutase activities were unchanged. Glutathione peroxidase activity was diminished. In the pancreas, only glutathione peroxidase activity was affected, and it was reduced in both the BHA and BHT treatment groups. Although the pancreas is refractory to the enzyme inductive effects of these antioxidants, morphometric analysis of foci demonstrated chemoprevention by BHA and BHT of azaserine-induced foci. Whether this reduction reflected inhibition of an initiation, postinitiation , or a combination of effects was not known.     

Different modifying response of butylated hydroxyanisole, butylated hydroxytoluene, and other antioxidants in N,N-dibutylnitrosamine esophagus and forestomach carcinogenesis of rats

The modifying effects of antioxidants were examined in a carcinogenesis system after N,N-dibutylnitrosamine treatment. Male F344 rats were given 0.05% N,N-dibutylnitrosamine in their drinking water for 4 wk and then treated with basal diet containing 2% butylated hydroxyanisole (BHA), 1% butylated hydroxytoluene (BHT) with 7 ppm vitamin K, 0.8% ethoxyquin, 5% sodium L-ascorbate, 5% sodium erythorbate, or no added chemical for 32 wk. BHA enhanced forestomach carcinogenesis but did not enhance esophageal carcinogenesis. BHT enhanced esophageal carcinogenesis but did not enhance forestomach carcinogenesis. Ethoxyquin significantly enhanced esophageal tumorigenesis. Neither esophageal nor forestomach carcinogenesis was affected by the other antioxidants evaluated. BHA significantly increased DNA synthesis of the forestomach epithelium, whereas BHT tended to increase that of the esophageal epithelium. Thus, BHA and BHT showed different modifying responses in carcinogenesis of the esophagus and forestomach.     

Inhibition of binding of 2-acetylaminofluorene to DNA by butylated hydroxytoluene and butylated hydroxyanisole in vitro

Numerous studies have shown that the food antioxidants butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), under specific exposure conditions, can inhibit hepatocarcinogenesis induced by various carcinogens. The purpose of the present work was to study the biochemical mechanisms responsible for the anticarcinogenic activity of BHA and BHT using in vitro systems. The effects of BHA and BHT on the binding of 2-acetylaminofluorene (2-AAF) to DNA was determined in a microsomal system and in primary cultures of rat hepatocytes. It was found that both antioxidants reduce the binding of 2-AAF and that of N-OH-2-acetylaminofluorene (N-OH-2-AAF) to calf thymus DNA in the presence of liver microsomes. The inhibition was however more pronounced with the parent compound. Lower levels of DNA binding were also detected in hepatocytes incubated with 2-AAF along with BHA or BHT. These results suggest that phenolic antioxidants can exert anticarcinogenic activity through modulation of carcinogen interaction with DNA which may reflect on alteration in carcinogen metabolic activation.     

Histologic and autoradiographic studies on the forestomach of hamsters treated with 2-tert-butylated hydroxyanisole, 3-tert-butylated hydroxyanisole, crude butylated hydroxyanisole, or butylated hydroxytoluene

The inductions of hyperplasia and neoplastic lesions in the forestomach of Syrian golden hamsters by 2-tert-butylated hydroxyanisole [(2-tert-BHA) CAS: 121-00-6], 3-tert-butylated hydroxyanisole [(3-tert-BHA) CAS: 88-32-4], crude butylated hydroxyanisole [(BHA) CAS: 25013-16-5], and butylated hydroxytoluene [(BHT) CAS: 128-37-0] were compared histopathologically and autoradiographically. In hamsters fed the 2-tert-BHA diet, severe hyperplasia developed from week 4, reaching a maximum level in week 16 of 0.56 cm/10 cm basement membrane (bm), and papillomatous lesions appeared in week 16 (0.13 cm/10 cm bm). In hamsters fed 3-tert-BHA or crude BHA, severe hyperplasia developed from week 1, which reached a maximum level in week 4 of 3.63 cm/10 cm bm with 3-tert-BHA and 5.10 cm/10 with crude BHA; it then decreased. Papillomatous lesions were found in week 3 in hamsters fed 3-tert-BHA and in week 4 in hamsters fed crude BHA; they increased to maximum levels in week 16 of 0.50 cm/10 cm bm with 3-tert-BHA and 0.29 cm/10 cm bm with crude BHA. Mild hyperplasia occurred slightly more often in hamsters fed the BHT diet than in the control group. BHT induced no severe hyperplasia and papillomatous lesions. Changes in the labeling index of the forestomach epithelium paralleled the histologic changes, except in hamsters fed the BHT diet in which no significant increase in the labeling index was observed throughout the experiment. These data suggest that the tumorigenic action of crude BHA on hamster forestomach is largely due to 3-tert-BHA and that BHT does not induce forestomach tumors in hamsters.     

Initiating potential of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and 3,3',4',5,7-pentahydroxyflavone (quercetin) in two-stage mouse skin carcinogenesis

Tumor initiating activity of 3,3',4',5,7-pentahydroxyflavone (quercetin), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and 2-(2-furyl)-3-(5-nitro-2-furyl) (AF-2) acrylamide) were tested in a two-stage mouse skin carcinogenesis model using 12-O-tetradecanoyl phorbol-13-acetate (TPA) as a promoter. These compounds dissolved in dimethyl sulfoxide were topically applied twice weekly for 5 weeks on the dorsal skin, and then followed by TPA for 47 weeks. The total initiating dose was 100 mg for each compound. 7,12-Dimethylbenz(a)anthracene (DMBA) at a total dose of 100 micrograms was used as a positive control compound. AF-2 induced skin tumors in 35% of the mice (average of 0.4 tumors/mouse), HBA in 15% in (0.2/mouse), BHT in 13% (0.13/mouse) and quercetin in 5% (0.1/mouse). No tumors appeared in the groups treated with either test chemicals alone or TPA alone. Statistical analysis according to either Fisher's exact test or Peto's trend test revealed significant differences for tumor appearance in the AF-2/TPA and BHA/TPA followed by TPA groups as compared to in the DMSO/TPA group. The results indicate that AF-2 and BHA have weak tumor initiating activity on mouse skin, but such effects are not apparent for BHT or quercetin.     

Inhibition by dehydroepiandrosterone of butylated hydroxyanisole (BHA) promotion of rat-bladder carcinogenesis and enhancement of BHA-induced forestomach hyperplasia

The effects of dehydroepiandrosterone (DHEA) with/without ribonucleoside (RN_s) supplementation on butylated hydroxyanisole (BHA) bladder-tumor promotion and forestomach carcinogenesis were investigated. Male F344 rats were given N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in their drinking water for 4 weeks and then received basal diet or diet containing BHA, DHEA, a mixture of RN_s, BHA + DHEA or BHA + DHEA + RN_s for 32 weeks. The occurrences of papillomas and carcinomas in the urinary bladder were increased in the groups given BHA or BHA + DHEA + RN_s, as compared with control group values. In comparison with the BHA group, the BHA + DHEA group incidences and numbers of these tumors were decreased. However, the incidence and multiplicity of papillomas in the group given BHA + DHEA + RN_s were again elevated. DNA synthesis levels in normal-appearing bladder epithelium, but not tumor cells, were closely correlated with the observed level of promotion in most groups. The case of DHEA alone proved exceptional in that DNA synthesis was markedly decreased without any significant influence on lesion development. In the forestomach, DHEA, which itself was associated with slight although non-significant hyperplasia, enhanced BHA-induced epithelial lesions, characterized by marked basal-cell proliferation and keratin-cyst formation, independently of additional RN_s administration. Our results suggest that the anti-promoting effects of DHEA in the bladder depend on a deficiency in the pentose phosphates necessary for production of nucleosides. Organ-specific modulation is indicated by the enhancing effects of DHEA on BHA-induced forestomach hyperplasia.     

Inhibition of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinogenesis by concomitant or postcarcinogen antioxidant exposure

When administered prior to or at the time of carcinogen exposure, the phenolic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are effective inhibitors of carcinogenesis in several target organs. However, chronic, postcarcinogen administration of BHT apparently enhances tumorigenesis in certain animal models for liver and lung cancer. The present study was performed to determine the effects of BHA and BHT on mammary carcinogenesis when antioxidant exposure is limited to defined periods encompassing or following carcinogen availability. At 50 days of age (Time O), virgin female Sprague-Dawley rats (25/group) were given a single intragastric dose of 8 mg of 7,12-dimethylbenz(a) athracene . Basal diet (Wayne Lab Meal) was supplemented with 5000 or 2500 mg of BHA or BHT/kg by the following protocol: 2 weeks before until 1 week after carcinogen administration; 1 week after carcinogen administration until the end of the study; or none. The experiment was terminated 210 days after 7,12-dimethylbenz(a)anthracene administration, and all mammary tumors were confirmed histologically. When administered by the 2 weeks before to 1 week after schedule, both BHA and BHT were effective inhibitors of mammary carcinogenesis. However, the compounds also were active in chemoprevention when administered by the 1 week after to end protocol. These data indicate that the anticarcinogenic activity of antioxidants is not limited to influences on carcinogen metabolism, since both BHA and BHT inhibited mammary tumor induction when their administration was begun following clearance of the carcinogen from the mammary gland. The anticarcinogenic activity of postcarcinogen administration of BHA and BHT in the mammary gland is in contrast to the apparent tumor-enhancing activity of BHT in the liver and lung.     

Lack of Promoting Effects of α-Linolenic, Linoleic or Palmitic Acid on Urinary Bladder Carcinogenesis in Rats

Potential promoting effects of α-linolenic, linoleic and palmitic acids were investigated in a two-stage urinary bladder carcinogenesis model. In experiment 1, male F344 rats were given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosainine (BBN) in their drinking water for 4 weeks and then basal diet containing 10%α-linolenic, 10% linoleic or 10% palmitic acid along with 0.2% butylated hydroxyanisole (BHA) as an antioxidant for 24 weeks. The development of tumors in the urinary bladder was not increased by treatment with any of the fatty acids. In experiment 2, male F344 rats were given 10%α-linolenic, 10% linoleic or 10% palmitic acid along with 0.2% BHA in their diet for 8 weeks without prior BBN treatment. The administration of fatty acids was not associated with any increase in the 5-bromo-2'-deoxyuridine labeling index of the urinary bladder epithelium. Serum and/or urine fatty acid Ievels increased in the cases of α-linolenic and linoleic acid treatments, but not with palmitic acid. Under the present experimental conditions neither the two polyunsaturated nor the one saturated fatty acid exerted any promoting effect on urinary bladder carcinogenesis.     

Modification of N-methyl-N-nitrosourea initiated carcinogenesis in the rat by subsequent treatment with antioxidants, phenobarbital and ethinyl estradiol

The modifying effects of butylated hydroxyanisole (BHA), sodium L-ascorbate (SA), phenobarbital (PB) and ethinyl estradiol (EE) were studied by their administration to F344 rats subsequent to initiation with N-methyl-N-nitrosourea (MNU), a wide-spectrum carcinogen inducing tumors in many organs. Rats were initially given 4 doses of MNU (50 mg/kg) intraperitoneally within a 2-week period and then placed on a diet containing BHA (1%), SA (5%), PB (0.05%) or EE (0.001%) for 23 weeks prior to killing. Since the experiment was based on a whole body concept of carcinogenesis, all major organs were examined histologically and histochemically for any preneoplastic lesions. BHA enhanced forestomach and urinary bladder carcinogenesis as did SA also for the urinary bladder, whereas PB enhanced the induction of gamma-glutamyl transpeptidase positive (gamma-GT+) foci in the liver and also the incidence of thyroid carcinoma and forestomach carcinoma. In contrast, EE inhibited the induction of thyroid tumors, malignant lymphoma or leukemia. Thus these compounds, when given after initiation of many organs by a single carcinogen, exert an influence on the site of tumor development by, as yet unknown, organotropic modifying effects.     

Dose-related inhibition of aflatoxin B1 induced hepatocarcinogenesis by the phenolic antioxidants, butylated hydroxyanisole and butylated hydroxytoluene

The effect of butylated hydroxyanisole (BHA) or butylated hydroxytoluene(BHT) on the carcinogenicity in rats of aflatoxin B₁ (AFB₁)was investigated. AFB₁ was administered by gastric intubationto male F344 rats at 25µg/kg body wt three times a weeksuch that a total dose of 1.5 mg/kg (0.48mmol/kg) body wt wasgiven over a period of 20 weeks and diets containing either1000 or 6000 p.p.m. BHA or BHT were fed starting one more weekbefore carcinogen, during administration and for one week aftercessation. Animals were killed during exposure and at intervalsup to 24 weeks after cessation. Liver altered foci and neoplasmswere quantified using the exclusion of cellular iron after iron-loadingand -glutamyl transpeptidase reaction, as well as conventionalstaining for identification. Exposure to AFB₁ alone inducedsubstancial numbers of altered foci after 20 weeks and at 24weeks after cessation of exposure, the incidence of hepato-cellularneoplasms was 63%. In the groups receiving BHA or BHT togetherwith AFB₁ the numbers of altered foci were decreased at alltime points and at termination, the final incidence of livercell neoplasms and number of neoplasms peranimal were also reducedin a dose-related manner. Neoplasms in other organs were rareand were not affected by antioxidant treatment, except for apossible reduction of colon cancer. Thus, BHA and BHT inhibitedthe hepatocarcino-genesis of concurrently administered AFB₁without shifting the organotropism.     

Effect of retinoic acid, butylated hydroxytoluene, selenium and sorbic acid on azo-dye hepatocarcinogenesis

Groups of male Sprague-Dawley rats were maintained on a basal diet containing 0.05% 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) for 9 weeks. The diets of these groups were supplemented at certain stages of the study with retinoic acid, butylated hydroxytoluene (BHT), or sorbic acid. In other groups, selenium (as sodium selenite) was added to the drinking water. The study was terminated after 9 weeks and the livers evaluated for pre-cancerous changes and presence of tumors. 38/42 animals in the control groups given the diet containing the 3'-MeDAB developed liver tumors. Only 3/27 rats given the 3'-MeDAB regimen supplemented with retinoic acid had liver tumors. A similar reduction was obtained with BHT, while sorbic acid exerted no protective effect against hepatocarcinogenesis. Se supplementation afforded some protection if given throughout or during the early stages of azo-dye administration and a lesser effect if given during the later stages of dye feeding.     

Dose-dependent reduction of N-2-fluorenylacetamide-induced liver cancer and enhancement of bladder cancer in rats by butylated hydroxytoluene

The modifying effect of four dose levels of butylated hydroxytoluene (BHT) on liver and bladder carcinogenesis by N-2-fluorenylacetamide (FAA) was studied in rats. FAA (200 ppm) was fed simultaneously with four levels of BHT (300, 1000, 3000, and 6000 ppm) to male F344 Fischer strain rats. Groups of animals were killed at 6, 12, 18, and 25 weeks. Hepatocellular-altered foci in the liver were identified histochemically by the exclusion of cellular iron after iron loading and by reaction for gamma-glutamyltranspeptidase. FAA alone induced altered hepatocellular foci which increased in number with duration of exposure, and by 25 weeks of feeding, 100% of rats had liver neoplasms. Concurrent feeding of BHT produced a dose-dependent reduction in the numbers of altered foci and the areas of liver sections occupied by histochemically altered cells, as well as a reduction in the incidence of liver neoplasms and the number of neoplasms per animal. These findings indicate that reduction of the number of foci at an early stage is predictive of reduced incidence of neoplasms at a late stage. Thus, BHT inhibited in a dose-dependent manner the hepatocarcinogenesis of concurrently fed FAA. However, although no bladder neoplasms occurred in rats given either BHT or FAA alone, groups fed BHT and FAA together developed bladder neoplasms in proportion to the dose of BHT. The effect of BHT on both liver and bladder carcinogenesis could be explained by BHT causing an alteration of the metabolism of FAA in the liver, resulting in less activation and greater urinary excretion of carcinogenic metabolites. In addition, BHT at high dose levels may exert a promoting effect on bladder carcinogenesis. Accordingly, it is suggested that the chemopreventive effect of phenolic antioxidants should be investigated with attention to the possibility that they not only inhibit carcinogenesis in the main target organs but also modify carcinogenesis in other organs.     

Enhancing Potential of 6 Different Carcinogens on Multi-organ Tumorigenesis after Initial Treatment with N-Methyl-N-nitrosourea in Rats

The advantages of applying a whole-body concept to the assessment of carcinogenic potential of compounds in a two-stage model after initiation by N-methyl-N-nitrosourea (MNU) were investigated. Male, 6-week-old F344 rats were injected with MNU (20 mg/kg, i. p.) twice a week for 4 weeks and they then received 3,2'-dimethyl-4-aminobiphenyl (DMAB) (50 mg/kg, s.c., once a week), N,N'-dibutylnitrosaraine (DBN) (0.05%, in drinking water), N-bis(2-hydroxypropyl)nitrosamine (DHPN) (0.1%, in drinking water), diethylstilbestrol (DES) (2.5 ppm, in diet), sodium o -phenylphenate (S. OPP) (2%, in diet) or captafol (0.15%, in diet) for 20 weeks. All six carcinogens enhanced the incidences of preneoplastic and neoplastic lesions in their respective target organs: liver, pancreas, small intestine and urinary bladder with DMAB; liver, esophagus, forestomach and urinary bladder with DBN; thyroid, lung, liver, esophagus, forestomach, small intestine and urinary bladder with DHPN; liver and forestomach with DES; and thyroid, forestomach, kidney and urinary bladder with S. OPP; liver and forestomach with captafol. The results suggested that prior treatment with MNU sensitized the tissues to the organotropic carcinogenic potential of chemicals given thereafter for as short a period as 20 weeks. Thus, this system could be utilized as a whole-body medium-term bioassay system for the screening of environmental carcinogens, bridging the gap between in vitro mutagenicity and long-term carcinogenicity tests.