Short-term pathological and proliferative effects of butylated hydroxyanisole and other phenolic anti-oxidants in the forestomach of Fischer 344 rats

Food grade butylated hydroxyanisole (BHA) when incorporated in the diet and fed to male Fischer 344 rats for 9 or 27 days induced proliferative squamous epithelial changes in the lesser curvature of the forestomach proximate to the glandular stomach. These changes were assessed histopathologically and by [methyl-³H]thymidine radioautography. It was shown that BHA mixed dry into powdered diet, incorporated into the diet in corn oil, or in a pelleted diet, induced similar effects. When levels of 2%, 1%, 0.5%, 0.25%, 0.1% and 0% BHA were incorporated in rat diet for 9 days the proliferative effect appeared to show a no effect level at 0.25% based on the [methyl-³H]thymidine-labelling index. Other food use antioxidants, namely butylated hydroxytoluene or tertiary butylhydroquinone, induced a lesser response that BHA at the maximum dose employed in the study. Propyl gallate was without effect. Propyyl-4-hydroxybenzoate, a food use phenol, on the other hand, induced a less pronounced response than BHA but was more effective than the other antioxidants. Because increased cellular proliferation often provides an optimal milieu for tumor formation, it is suggested that these observation may ne relevant to rat forestomach tumors induced by BHA.     

Metabolism of 2- and 3-tert-butyl-4-hydroxyanisole (2- and 3-BHA) in the rat. (II): Metabolism in forestomach and covalent binding to tissue macromolecules

The mechanism of action of 2(3)-tert-butyl-4-hydroxyanisole (2-BHA or 3-BHA) on rat forestomach epithelium was studied by examining the metabolites of BHA in the stomach and the covalent binding of BHA to macromolecules in the forestomach epithelium. Male F344 rats 6 weeks old were given a single intragastric injection of 1 g/kg body wt of [tert-14C]-3-BHA (Bu-3-BHA) or [methyl-14C]-3-BHA (Me-3-BHA), and 6 h later BHA metabolites in the forestomach, glandular stomach and stomach contents were examined by thin-layer chromatography. No significant amounts of metabolites were detected in the forestomach or glandular stomach epithelium and almost all the radioactivity in these tissues was extracted with organic solvents. In in vitro experiments also, no significant amounts of metabolites were detected when the 9000 g supernatant of the forestomach or glandular stomach epithelium, or gastric juice was incubated with Bu-3-BHA in the absence or presence of NADPH. In binding studies, rats were given Bu-3-BHA, [tert-14C]-2-BHA (Bu-2-BHA), Me-3-BHA or [methyl-14C] butylated hydroxytoluene (Me-BHT) intragastrically at a dose of 1 g/kg body wt with or without pretreatment with unlabelled 1% 3-BHA or BHT in the diet for 6 days. Six hours after treatment with a labelled compound, the rats were sacrificed and the DNA, RNA and protein of their forestomach, glandular stomach, liver and kidney were isolated. Bu-3-BHA, Bu-2-BHA and Me-3-BHA did not bind covalently to forestomach DNA or RNA, and the amounts of radioactivity of these compounds bound to proteins in the 4 tissues were similar. These findings suggest that BHA acts on the forestomach epithelium directly without metabolic activation, and that its action is not related to its binding to DNA or RNA.     

A carcinogenesis reversibility study of the effects of butylated hydroxyanisole on the forestomach and urinary bladder in male Fischer 344 rats

A reversibility study was initiated to determine if the length of feeding with 2% butylated hydroxyanisole (BHA) altered the incidence of forestomach lesions observed after a 24-month observation period. Groups of male Fischer 344 rats were fed 2% BHA for 0, 3, 6, 12, and 24 months and then the basal diet for the completion of the 24-month experimental period. Subgroups were serially sacrificed for histopathological examination and [methyl-3H]thymidine radioautography at the time when each group of animals was transferred to the basal diet and also at 15 months. The results showed that except for carcinomas and some epithelial downgrowths, cellular proliferation, measured by radioautography in the epithelium lining the greater and the lesser curvature of the forestomach, remained dependent on the continuous presence of 2% BHA for, at least, 12 months. Superficial hyperplasias, inflammatory lesions and many of the papillomas regressed after cessation of treatment at 12 months. The epithelial downgrowths did not appear to enlarge after the BHA was withdrawn. The squamous cell carcinomas occurred in almost identical yields whether the rats were fed 2% BHA for 12 months and then returned to the basal diet for 12 months or received 2% BHA continuously for 24 months. It is shown here that at several times, 2% BHA stimulated the [methyl-3H]thymidine labelling index of the transitional epithelium of the urinary bladder and that at 3 months the no observed effect level was greater than 0.5% BHA. The significance of the studies on the forestomach and bladder epithelia are discussed. It is concluded that the lesions induced by BHA are most unlikely to be relevant to humans exposed to much lower levels of BHA.     

Butylated hydroxyanisole (BHA) does not cause forestomach hyperplasia by inhibiting the release of gastric mucus

High doses of BHA cause hyperplasia and subsequent neoplasia in the rodent forestomach and can inhibit gastric prostaglandin (PG) synthesis in vitro. This paper examines the hypothesis that BHA induced forestomach hyperplasia occurs in response to a reduction of gastric mucus, with consequent irritation of the forestomach. This could result from inhibition of the formation of the PG's which mediate the synthesis and release of protective mucus. Groups of 10 rats received 0 or 2% BHA in the diet for 1 or 3 weeks and a positive control group was fed a diet containing indomethacin (3.5 mg/kg), a potent inhibitor of PG synthesis. After 1 week BHA caused focal erosion and ulceration of the forestomach consistent with an irritant effect, but 2 weeks later the epithelium was healed, thickened and markedly hyperplastic. Histochemical staining for mucus showed that the development of forestomach hyperplasia was associated with increased amounts of gastric and duodenal mucus and increased numbers of serotonergic-cells in the gastric and duodenal epithelium. In contrast, indomethacin caused a marked reduction in both gastric and Brunner's gland mucus. Neither BHA nor indomethacin exerted an effect on one specific type of mucus (viz: neutral, acidic or mixed) in the stomach. These results do not support the hypothesis that forestomach hyperplasia arises from an inhibition of either the synthesis or release of gastric mucus. It is possible that the increased numbers of serotonergic-cells are related to the initial ulcerative, or subsequent hyperplastic response.     

Effects of BHA and related phenols on the forestomach of rats

To determine the pathogenesis of BHA-induced forestomach lesions, the nature and time course of the early lesions in the forestomach of Wistar rats were studied. The rats were given BHA at a dose level of 2% in a powdered diet or by oral intubation of 1 g BHA/kg body weight/day in arachis oil. The hyperplastic changes in the mucosa were visible 1 day after the second application. The localization was dependent on the mode of application. Dietary exposure yielded changes in the area of the limiting ridge; oral intubation of BHA produced lesions in the apex of the forestomach. In a subchronic 90-day feeding study in rats, no recognizable effect was observed when 0.125% BHA was incorporated into the diet as a solution in arachis oil. In reversibility studies, severe forestomach lesions observed after feeding 2% BHA for 6, 12 or 15 months regressed almost completely following withdrawal of the BHA for a period of 7 months. BHA induced similar forestomach damage in NMRI mice and Syrian golden hamsters, whereas guinea-pigs, a species having no forestomach, did not show comparable lesions. Substances with similar chemical structure were tested in short-term feeding studies (tert-butylhydroquinone, 4-methoxyphenol, 1,4-dimethoxybenzene, hydroquinone, 3-methoxyphenol, 2-methoxyphenol, anisole, p-cresol, phenol and BHT). Only 4-methoxyphenol strongly affected the forestomach mucosa in a manner similar to that associated with BHA. The methoxy group in the para position seems to be important for the hyperplasiogenic activity.     

Pathology of BHA- and BHT-induced lesions

The pathology lesions from three studies, two with butylated hydroxyanisole (BHA) and one with butylated hydroxytoluene (BHT), are reviewed. When BHA was fed at 0.5 and 2.0% of the diet to F344 rats for two years, there was an increase in epithelial hyperplasia of the forestomach at both treatment levels. Papilloma and squamous-cell carcinoma of the forestomach were increased at the 2.0% level. When BHA was fed to beagle dogs at 1.0 and 1.3% of the diet for 180 days, no lesions/tumours of the distal oesophagus or stomach could be identified either at gross necropsy or by light or electron microscopy. The BHT was fed to Wistar rats at 0, 25, 100 and 250 mg/kg body weight. At the highest dose there was an increase in the number of rats with hepatocellular adenoma and with hepatocellular carcinoma.     

Biological effects of short-term feeding to rats of repeatedly used deep-frying fats in relation to fat mutagen content

The effects of short-term feeding of mutagen containing, heated deep-frying oils on urinary and faecal mutagenicity, plasma clinical biochemical parameters, peroxidative effects and cell proliferative indices in the gastro-intestinal tract were determined in rats. Repeatedly used frying oils [a saturated fatty acid-rich coconut oil (CO) and a polyunsaturated fatty acid (PUFA)-rich (greater than 60% PUFA) vegetable frying oil (PO)] were administered to groups of seven rats at a level of 10% (by weight) in the diet for 4 wk; control groups were fed equal amounts of the unheated oils. Both heated oils showed direct-acting mutagenicity to Salmonella tester strain TA97; heated PO was also mutagenic to strain TA100. Both heated CO and heated PO contained detectable amounts of thiobarbituric acid-reactive substances (TBA-RS). In heated PO, hydroperoxides of linoleic acid were also present. In groups fed heated oils the mutagenicity of urine and faeces to strain TA97 was not found to be increased in comparison with the control groups. Faecal mutagenicity to strain TA100 was also unaffected by consumption of heated oils. Urinary excretion of TA100 mutagens was significantly increased in rats fed heated PO. Plasma alkaline phosphatase activity was clearly raised in rats fed heated PO, in comparison with rats fed unheated oils or heated CO. In addition, other clinical biochemical plasma parameters showed a tendency to be increased in rats fed heated PO, indicating hepatic and renal cellular toxicity. Urinary and faecal excretion of thiobarbituric acid-reactive substances (TBA-RS) were also slightly, but not significantly, increased in rats fed heated PO. Feeding heated CO to rats did not result in increased plasma enzyme activities and excretion of TBA-RS, nor in increased cell proliferation in gastro-intestinal tissues. Cell proliferation of the oesophageal tissues were slightly, but significantly, increased in rats fed heated PO, in comparison with the group fed unheated PO. Tissues of the glandular stomach and colon/rectum did not show significantly enhanced cell proliferation in the group fed heated PO. The results obtained in this study indicated that consumption of heated oils containing TA100 mutagens and oxidation products of linoleic acid produced indications of cellular damage to liver and kidneys, and increased urine mutagenicity, as well as enhanced cell proliferation in the oesophagus.     

A 12-week study of BHA in the cynomolgus monkey

Butylated hydroxyanisole (BHA) given by gavage to female cynomolgus monkeys on 5 days/wk for 84 days produced transient changes in selected serum chemistry and haematology parameters. Terminal observations revealed increased liver size, decreased hepatic monooxygenase activity and an increase in the mitotic index of the oesophageal epithelium. Several of these observations are similar to those reported for rodents also given BHA at or near the maximum tolerated dose. Gastroscopic evaluation of the stomach and oesophagus at monthly intervals and extensive gross and histopathological examination failed to reveal the proliferative effects seen in the forestomach of rats fed diets containing BHA.     

Safety evaluation of butylated hydroxyanisole from the perspective of effects on forestomach and oesophageal squamous epithelium

Butylated hydroxyanisole (BHA) induces tumours of the squamous epithelium of the forestomach of rodents, but not at other sites. Although humans do not have squamous epithelium in their stomach the likelihood that BHA will induce tumours of the squamous epithelium of the oesophagus needs to be considered. Studies in several species indicate that the forestomach epithelium is very responsive to hyperplastic and neoplastic change induced by BHA, but the oesophageal epithelium is not responsive. The lack of effect in the oesophagus is likely to be due to the fact that the rapid speed of transit through the oesophagus limits the exposure time of the oesophageal mucosa to the food contents. Conversely, as the rodent's forestomach has storage function, exposure of the squamous epithelium of the forestomach would be continuous. The fact that the no-observed-effect level for hyperplasia of the oesophageal mucosa is several hundred times the acceptable daily intake for BHA supports the view that BHA would not be a human carcinogen at food additive levels of use.     

Studies on antioxidants: their carcinogenic and modifying effects on chemical carcinogenesis

Studies were conducted on the carcinogenic activity of butylated hydroxyanisole (BHA) in rats and hamsters. To obtain information concerning the mechanism of action of BHA on the forestomach, the following areas were examined: the effects of 12 phenolic compounds structurally related to BHA on the hamster forestomach, the effects of combinations of BHA and other antioxidants on the rat forestomach, and the metabolism of BHA in the forestomach. Also examined were the effects of several antioxidants on two-stage carcinogenesis in rats. Squamous-cell carcinomas were induced in the forestomach of rats and hamsters fed BHA. In a limited study, 1 of 13 hamsters developed a squamous-cell carcinoma. The tumorigenic action of crude BHA on the forestomach was largely due to the action of 3-tert-BHA. p-tert-Butylphenol and 2-tert-butyl-4-methylphenol induced pronounced hyperplasia and papillomas in the hamster forestomach. BHA and other antioxidants, particularly propyl gallate and ethoxyquin, showed additive effects in inducing forestomach hyperplasia and cytotoxicity. Neither BHA nor its metabolites were found in the forestomach epithelium, although small amounts of metabolites were detected in the stomach contents. Thus, a direct action on the stomach epithelium may be exerted by BHA itself or by metabolites formed on interaction of BHA with gastric juice. BHA enhanced forestomach carcinogenesis initiated in rats by N-methyl-N'-nitro-N-nitrosoguanidine or N-methylnitrosourea (MNU) and enhanced urinary bladder carcinogenesis initiated by MNU or N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). In contrast, it inhibited carcinogenesis initiated in the liver by either diethylnitrosamine or N-ethyl-N-hydroxyethylnitrosamine (EHEN) and mammary carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene (DMBA). BHT promoted urinary bladder carcinogenesis initiated by BBN or MNU and thyroid carcinogenesis initiated by MNU, but inhibited ear-duct carcinogenesis initiated by DMBA. Ethoxyquin promoted EHEN-initiated kidney carcinogenesis, but inhibited both DMBA-initiated mammary and EHEN-initiated liver carcinogenesis. Sodium ascorbate promoted forestomach and urinary bladder carcinogenesis, and sodium erythorbate also enhanced urinary bladder carcinogenesis. alpha-Tocopherol inhibited ear-duct carcinogenesis. No antioxidants tested had any effect on glandular stomach carcinogenesis. Thus antioxidants have independent modifying (promoting or inhibitory) effects in different organs.     

BHA study in pigs

Butylated hydroxyanisole (BHA) was given to pregnant SPF pigs (Danish Landrace) in doses of 0, 50, 200 and 400 mg/kg body weight/day from mating to day 110 of the gestation period. The BHA was mixed in the diet (pelleted). Caesarean section was performed on gestation day 110. BHA affected neither the reproduction data nor the incidence of defects in the foetuses. Significantly lower weight gain was observed in the group of dams on the highest dose. Absolute and relative organ weights for the liver and thyroid gland showed a dose-related increase. Proliferative and parakeratotic proliferative changes of the stratified epithelium of the stomach were found in both control and treated pigs. In addition, proliferative and parakeratotic changes of the oesophageal epithelium were observed in a few pigs in the two groups on the highest doses. Papillomas were not found, and no changes of the glandular part of the stomach were observed.     

Metabolism of 2- and 3-tert-butyl-4-hydroxyanisole (2- and 3-BHA) in the rat (I): Excretion of BHA in urine, feces and expired air and distribution of BHA in the main organs

The mechanism of the carcinogenic or toxic action of BHA on rat forestomach was examined by studies on the excretion and tissue distribution of radioactivity in F344 male rats given tert-butyl- or methoxy-labelled 3-BHA orally. Within 2 days after a single oral dose of labelled BHA at 1 g/kg body wt, 87-96% of the 14C was excreted, mainly in the urine with smaller amounts in the feces and expired air. More 14C was found in the tissues of rats given the methoxy-labelled compounds. The distributions of 14C in the forestomach and the glandular stomach were similar. At 168 h after treatment, more 14C was found in the forestomach of rats given 2-BHA than in that of rats given 3-BHA. These results indicate that excretion of BHA is rapid, that 4-O-methyl demethylation may take place readily and that demethylated methyl group may become distributed non-specifically in tissues. The carcinogenic or toxic action of BHA on the forestomach does not seem to be due accumulation of BHA in the forestomach.     

Evaluation of the oral toxicity of acetaldehyde and formaldehyde in a 4-week drinking-water study in rats

A subacute oral toxicity study of acetaldehyde and formaldehyde was carried out in rats. Groups of ten male and ten female 5-wk-old rats received one of the aldehydes in the drinking-water for a period of 4 wk, acetaldehyde being given at dose levels of 25, 125 and 675 mg/kg body weight/day and formaldehyde at dose levels of 5, 25 and 125 mg/kg body weight/day. A group of 20 males and 20 females served as controls and received unsupplemented drinking-water ad lib. An additional group of ten males and ten females was given unsupplemented drinking-water in an amount equal to the amount of liquid consumed by the group given the top dose of formaldehyde. Food and liquid intake were decreased in the groups on the top dose of both acetaldehyde and formaldehyde. Hyperkeratosis of the forestomach, observed only in the top-dose rats, was the only adverse effect of acetaldehyde detected. Effects of formaldehyde, also observed only in the top-dose group, were yellow discoloration of the fur, decreased protein and albumin levels in the blood plasma, thickening of the limiting ridge and hyperkeratosis in the forestomach, and focal gastritis in the glandular stomach. It was concluded that in this study the no-observed-adverse-effect levels of acetaldehyde and formaldehyde were 125 and 25 mg/kg body weight/day, respectively.     

A 13-week feeding study of butylated hydroxyanisole: the subsequent regression of the induced lesions in male Fischer 344 rat forestomach epithelium

Feeding butylated hydroxyanisole (BHA) to male Fischer 344 rats at concentrations of 2, 0.5, 0.25, 0.1 and 0% for 13 weeks led to proliferative lesions developing in the forestomach epithelium of the 2%-treated rats but not in other groups. The [methyl-3H]thymidine labelling index was raised in the 2%- and 0.5%-treated groups and showed an apparent no observable effect level at 0.25%. Within 1 week after withdrawal of BHA the labelling indexes in all treated groups returned to near the values in the controls. The induced mucosal lesions, however, reverted more slowly and even after 9 weeks on the basal diet, the stratified squamous epithelium along the lesser curvature, was still slightly thicker than the control. There were multilayered basal cell processes in the lamina propria with connections to the basal cell layer. The possible significance of these results to the ultimate development of cancer is discussed.     

The absorption and excretion of butylated hydroxyanisole in beagle dogs

Recent studies have indicated that administration of [14C]butylated hydroxyanisole (BHA) to rats, either orally or by intraperitoneal (i.p.) injection, resulted in high retention of radioactivity in the forestomach. The present study was undertaken to investigate the fate of [14C]BHA in non-rodents. 2 Groups of 5-mth-old male beagle dogs were fed a diet containing either 3% or 0.03% BHA for 7 days, and were injected i.p. with 3-tert-[methyl-14C]butyl-4-hydroxyanisole (Amersham International) at a dose of 30 muCi/kg. On the 7th day after [14C]BHA injection, all dogs were killed after fasting overnight, and the liver, kidney, heart, fat tissue and stomach were collected for radioanalysis. An additional 3 beagles served as control group. The fate of BHA after the single i.p. injection of [14C]BHA was examined by the determination of 14C-radioactivity in whole body, blood, urine, feces and several tissues. Blood, urine and feces samples were collected daily for 7 days. Blood samples were collected at intervals for 24 h. BHA was rapidly taken up in the bloodstream, and 50-80% of the total radioactivity was recovered in the urine within 2 days. 15-30% Appeared in the feces within 2 days. The tissue distribution of radioactivity 7 days after [14C]BHA injection showed only a small portion remaining in the stomach (0.16-0.19% of dose/g), liver (0.3-1.7%) and other tissues (0.02%). The radio-activity was almost evenly distributed in the three parts of the stomach (cardia, corpus and pylorus). These findings are in contrast with the previous data in rats that BHA can accumulate in high concentrations in the forestomach.     

Short-term effects of various phenols and acids on the Fischer 344 male rat forestomach epithelium

A series of phenols and acids was fed to rats for 9 days to determine effects on the [methyl-3H]thymidine labelling index and the histological appearance of the forestomach. A variety of proliferative effects in the rat forestomach were observed which paralleled changes in the [methyl-3H] thymidine labelling index. The 3-tert-butyl isomer of butylated hydroxyanisole (BHA) was as effective as the food grade mixture. In the 4-hydroxybenzoic acid ester series, activity was not found either with the free acid or the methyl ester. With the ethyl, n-propyl and n-butyl esters activity in the perfundic region of the forestomach increased with alkyl chain length, the n-butyl ester being nearly as effective as BHA. In contrast, 4-methoxyyphenol and propionic acid demonstrated their greatest effects in the midregion of the forestomach, the action of propionic acid not being apparent until 21 or 27 days of treatment. It was also found that after a 9-day treatment involving coadministration of BHA and acetylsalicyclic acid, the overall effect of the antioxidant on the forestomach was greatly reduced.     

Effects of dietary broccoli and butylated hydroxyanisole on liver-mediated metabolism of benzo[a]pyrene

Groups of male Wistar rats were given either a basal diet or diets supplemented with 10 or 25% broccoli or 0.8% BHA. Liver fractions were assayed for cytochrome P-450, for aryl hydrocarbon hydroxylase (AHH), glutathione-S-transferase and epoxide hydrolase activities and for benzo[a]pyrene (BaP) metabolism. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of liver microsomes was also performed. Mean relative liver weight in the BHA group was significantly higher than that of the control and 10% broccoli groups but not significantly higher than that of the 25% broccoli group. Gel electrophoresis of liver microsomes indicated a diet-dependent variation in intensity in a band that corresponded in mol wt to those of certain cytochrome P-450s. Diet-dependent increases (20-90% over control) in cytochrome P-450 and in the activities of AHH, glutathione-S-transferase and epoxide hydrolase were observed in livers from rats given broccoli-supplemented diets. Except for AHH activity, such increases also occurred in the group fed BHA. Analysis of BaP metabolites revealed that the proportion of 4,5-diol formed relative to the major diols identified was unchanged in the broccoli- or BHA-treated groups relative to the control group. The proportion of 9,10-diol formed was unchanged in the broccoli-fed groups but was significantly higher in the BHA group than in the control group. The proportion of cis and trans-7,8-diol formed was unchanged in both broccoli-fed groups but was significantly lower in the BHA group. In comparison with the control group, the ratio of phenol I (comprising primarily 9-OH-BaP) to total phenols (primarily 9-OH and 3-OH) was significantly decreased by about 30% in the 25%-broccoli group and by about 70% in the BHA group. Qualitative differences in the phenol-II peak (comprising 3-OH and 7-OH phenols) were also observed between samples from the controls and those of 25%-broccoli- and BHA-fed rats. The implications of these findings are discussed with respect to the effects of broccoli and BHA on benzo[a]pyrene toxicity.     

Effects of butylated hydroxyanisole on glutathione S-transferase and catechol O-methyltransferase activities in Syrian golden hamsters

The effects of dietary butylated hydroxyanisole (BHA) on the enzyme activities of glutathione (GSH) S-transferase and catechol O-methyltransferase (COMT) in the forestomach, small intestinal mucosa, and liver of Syrian golden hamsters and ICR/Ha mice were examined. GSH S-transferase activity in the hamster tissues was not enhanced appreciably after 1 or 4 weeks of feeding diets containing various concentrations of BHA. In general, short term (1 week) feeding of diets containing BHA did not differ from longer term (4 weeks) feeding of the same diets. In the forestomach of hamsters, a positive dose response on the activity of GSH S-transferase was obtained with increasing concentration of BHA in the diet for 1 or 4 weeks. The maximum effect of dietary BHA in hamsters was observed in the forestomach after 1 week of feeding, which induced an increase in GSH S-transferase activity to twice that of the control level. The same induction effect, however, was not apparent in the liver or in the small intestinal mucosa. Dietary BHA, at all concentrations studied, did not elicit any significant change in the activity of the GSH S-transferase enzyme in these two tissues. While the increase of enzyme activity in the forestomach of ICR/Ha mice was similar to that observed in the forestomach of hamsters, the induction of GSH S-transferase activity in the liver and in the small intestinal mucosa of the two animal species was drastically different. In contrast to the lack of response to dietary BHA in the hamster tissues, the induction of increased enzyme activity in the liver and intestinal mucosa of ICR/Ha mice, after 1 week of 2% BHA feeding, was greater than 7 and 11 times that of control respectively. The ineffectiveness of BHA as an enzyme inducer in the hamster tissues was similar for the activity of COMT. The enzyme activity in all three hamster tissues examined did not change significantly as a result of BHA incorporation into the diet for 1 week. In contrast, the COMT activity in the forestomach and small intestinal mucosa of the mouse was increased with increasing concentration of dietary BHA. At 2% BHA, the enzyme activity in the two tissues was 3 and 2 times that of the control level, respectively, whereas the enzyme activity in the liver remained at control level. These findings suggest that the overall unresponsiveness of detoxifying enzyme systems in the Syrian golden hamsters may be critical in the formation of forestomach tumors caused by BHA.     

Nitrate reduction, gastro-intestinal pH and N-nitrosation in gnotobiotic and conventional rats

The reduction of nitrate in germ-free, gnotobiotic and conventional rats was investigated using blood methaemoglobin values as indicative of nitrite formation. Nitrate reduction was found to occur in the absence of a microbial flora, and throughout the experiment the blood content of methaemoglobin was higher in germ-free than in conventional rats. In vitro incubations of the gastric and small-intestinal mucosae of germ-free rats confirmed the presence of a heat-labile nitrate-reducing system. Measurement of the gastro-intestinal pH of germ-free and conventional rats revealed a generally higher pH value throughout the germ-free gastro-intestinal tract, with highly significant differences in the luminal pH of the forestomach, jejunum/ileum and caecum and a significant difference in the pH of the glandular stomach. Although some formation of N-nitrosoproline from proline and nitrate occurred in germ-free and gnotobiotic rats, nitrosation proceeded more readily in conventional rats. This effect may have been due to the lower gastric pH in the conventional rats although a more direct role for the flora cannot be discounted.     

Short term feeding of hexadienal to postnatal rats: effects on stomach aldehyde dehydrogenase

Since a number of food and food products contain 2,4-hexadienal (HX), an unsaturated aldehyde, human exposure to HX is likely. Long term HX feeding to rodents induces forestomach cancers. Aldehyde dehydrogenases (ALDH) are key enzymes in the stomach for the metabolism of aldehydes. We examined the effect of short term feeding of HX on ALDH activity using HX as the substrate (HXDH) in different tissues of the GI tract. Feeding HX at a dose of 200 mg/kg body weight/day to post-suckling rats for 5 days elevated HXDH activity in the forestomach and esophagus but not in the glandular stomach, liver, small intestine or kidney. The induction of HXDH by HX was evident at 12.5 mg/kg dose and showed a dose-dependence up to 200 mg/kg. Increase in HXDH was time dependent but detectable after the first feeding (1 day). A similar dose (200 mg/kg) of acetylaldehyde or ethanol had no effect on HXDH activity. The increase in HXDH level in the forestomach and esophagus was transient. HXDH activity returned to normal 4 days after withdrawal of HX. Zymograms of gastric HXDH isozyme patterns in control and HX-fed counterparts were similar but showed selective increase in two particular forms in the forestomach. It is possible that these isoforms metabolize HX differently resulting in an accumulation of potential carcinogenic metabolites within the forestomach.