Dose Of Butylated Hydroxytoluene Research Articles

The protective role of the food additive butylated hydroxytoluene in the pancreas of aflatoxicosed adult albino rats

Introduction Aflatoxins are poisonous by-products from the fungus Aspergillus, with aflatoxin B1 (AFB1) being the most toxic. Very high concentrations of aflatoxins are found in nutritive seeds like maize, nuts, cereal grains, and rice. They have been implicated in many gastrointestinal and hepatic diseases. Studies on their effects on the pancreas are limited. Phenolic antioxidants like butylated hydroxytoluene (BHT) were reported to be chemoprotective against aflatoxicosis in some animal models. Aim This work aimed to demonstrate the histopathological effects of chronic AFB1 toxicity on the exocrine and endocrine pancreas and evaluate the possible protective role of BHT in these structural changes. Materials and methods Thirty adult male albino rats were randomly divided into three groups: group I served as the control group; group II received 60 µg/kg of AFB1 orally three times weekly for 5 consecutive weeks; and group III received a concomitant dose of BHT (0.05 mg/kg) orally along with AFB1. After the rats had been sacrificed, pancreatic specimens were taken and processed for histological, immunohistochemical, and morphometric study. Results Both exocrine and endocrine pancreas of group II showed marked degenerative changes. Acinar cells exhibited decreased or almost absent basal basophilia and reduced content of zymogen granules in periodic acid Schiff-stained sections. Periductal and perivascular fibrosis was observed with dilatation and congestion of blood vessels and inflammatory cell infiltration. β-Cells were degenerated and expressed decreased insulin immunostaining. There was significant decrease in islet surface area, islet diameter, islet volume, and numerical density of β-cells compared with the control group. The pancreas of group III showed marked improvement compared with the aflatoxicosed group. Conclusion Treatment with AFB1 for 5 weeks has a direct toxic effect on the exocrine and endocrine pancreas that can ultimately affect its performance. The use of BHT markedly attenuates these effects and can be considered an effective chemoprotective agent against AFB1 toxicity.

Protective Effects of Copper (I)-Nicotinate Complex Against Aflatoxicosis

Aflatoxicosis mostly leads to serious clinical signs of malnutrition, growth retardation, and impairment of utilization of nutrients, immunosupression, liver diseases and cancer. Detoxification or prevention of such toxicity is interesting to researchers in change field. In pursuit for an approach, the synthetic copper(I)- nicotinate complex has been evaluated against the traditional Butylated hydroxy toluene (BHT) for possible protective effects as a food additive in induced aflatoxicosed animals. Induction of toxicity in adult rats by AFB1 60 µg/kg body weight divided on three times a week for consecutive five weeks resulted in marked hepatic necrosis, collagen fibers around portal tract and iron deposits as well as features of general cellular collapse and cirrhosis. The protective effects were tested on two animal groups by associate doses of BHT (0.05 mg / kg B.W.) in one group and copper (I) nicotinate complex (0.4 mg/kg B.W.) in the other. Results showed, in both groups, almost normal histological as well as biochemical markers especially those treated with the copper complex, which suggests that anti-inflammatory copper complex can be used as a protective agent against aflatoxicosis when used in doses resemble those used in pharmaceutical vitamin supplements.

Abstract 4801: Bronchoalveolar lavage fluid (BALF) from subchronic exposure to butylated-hydroxytoluene (BHT) inhibits gap junctional communication in a toll-like receptor 4-dependent manner.

Abstract Subchronic dosing of BHT in mice is a well-known model used to discretely study the tumor promotion stage of lung adenocarcinoma. Using this model, we have previously shown that functional toll-like receptor 4 (TLR4) is protective against inflammatory sequelae and primary tumor formation in the mouse lung. Gap junctional intercellular communication (GJIC) has been shown to regulate the signal transduction pathways of cells, and inhibition of this communication by toxicant exposure has been linked to dysregulation and eventual carcinogenic endpoints. As part of our research on the protective nature of TLR4 during tumor promotion, we utilized the BHT subchronic model (4 weekly i.p. doses of BHT) to examine the early effects on BALF in both BALB/c (BALB; Tlr4 sufficient) and C.C3H-Tlr4Lps-d mice (BALBLps-d-d; Tlr4 mutant). Analysis of the BALF 24 hours after the final dose for protein content and cytology was indicative of an inflammatory profile in all mice treated with BHT. Magnitude of response to BHT was highest in the BALBLps-d mice; the BALB appeared resistant. In an effort to further characterize the response of the pulmonary epithelium, the BALF samples were used to treat cultured C10 cells, a murine immortalized line derived from BALB/c alveolar type II cells, which are a precursor cell type for adenocarcinoma. Using the scrape-load dye transfer assay, a well-defined method to measure GJIC, we demonstrated marked inhibition of cellular communication after a 4 hour exposure to cell-free BALF that is both treatment and strain dependent. The direction and magnitude of effect is in parallel with that of the direct analysis of the BALF, i.e. we observed more inhibition in BALF from BALBLps-d compared to BALB mice. In addition, quantitative PCR (QPCR) analysis of C10 cells exposed to cell-free BALF for 24 hours indicates alterations in the transcription patterns in genes of innate immunity that are also treatment and strain dependent. These novel findings suggest endocrine signaling from the inflammatory milieu are complicit in the inhibition of GJIC during the promotion phase of carcinogenesis. Furthermore, these data establish a link between active gap junctions, tumor promotion, and the protective nature of TLR4 in an ex vivo model which will facilitate a mechanistic examination of these protective effects at the molecular level. Funded by ACS RSG-10-162-01-LIB (AKB). Citation Format: Thomas Hill, Carla-Maria Alexander, Alison K. Bauer. Bronchoalveolar lavage fluid (BALF) from subchronic exposure to butylated-hydroxytoluene (BHT) inhibits gap junctional communication in a toll-like receptor 4-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4801. doi:10.1158/1538-7445.AM2013-4801

Hepatic and associated response of rats to pregnancy, lactation and simultaneous treatment with butylated hydroxytoluene

This paper describes changes in the livers of rats fed diets containing butylated hydroxytoluene (BHT) over two generations in two separate studies. BHT did not produce tumours when tested for carcinogenicity in several studies by the conventional way. However, when BHT was given to rats in a two-generation carcinogenicity study, a high incidence of hepatic tumours was found in males but not in female rats of the F 1 generation. A sequential study has been carried out to gain an insight into this unexpected finding, paying particular attention to the perinatal period. In the dose-ranging study designed to assess the tolerance of rats to BHT, groups of male and female rats (F 0 generation) were fed diets calculated to deliver 0, 500, 750 and 1000 mg/kg body weight/day. Following a loading period of 5 wk the rats were mated. The BHT content of the diet was not adjusted during pregnancy and lactation. Owing to the normal increase in food consumption during lactation, intakes peaked at double the nominal value by 21 days after the birth of pups. At this time the pups (F 1) were weaned onto control diet and maintained on it for 4 wk. At birth, the body weights of pups from the BHT-treated dams were comparable to those of the controls but at weaning the body weights of the pups from all three dose levels were less than those of the controls. At the termination of the experiment (4 wk after weaning), the pups from BHT-treated dams still weighed less than those from untreated controls. In the main experiment the F 0 generation were fed 0, 25, 100 and 500 mg/kg body weight/day. Their offspring (F 1 generation) were weaned on diets containing the same amount of BHT as the respective parents, apart from the group given the highest dose level (500 mg/kg body weight/day). This dose level was reduced to 250 mg/kg body weight/day at weaning in order to conform with previously published findings. The pups from the dams given the highest dose level were maintained on a dietary concentration of 250 mg/kg body weight/day for the entire study. A group of age-matched non-pregnant females was also studied and the results obtained compared with those from pregnant dams. Pups from all groups were examined at day 20 of gestation, at weaning (21 days after birth), and at 4 and 22 wk post-weaning. There were no effects on fertility and no increase in foetal abnormalities at any dose of BHT. Dams receiving BHT at a nominal dose of 500 mg/kg body weight/day showed liver enlargement accompanied by induction of pentoxyresorufin O-depentylase and glutathione S-transferase, and proliferation of the endoplasmic reticulum. Pups from these dams were of the same weight at birth as controls but lost weight during the lactation period. This deficit was not recovered by the time the experiment was terminated. Hence, in two independent studies, the only significant finding in rats treated with BHT in utero and during lactation was that the weight gain of pups during lactation was less than expected when dams received at least 500 mg BHT/kg body weight/day. The body weight of pups did not return to normal following a return to a control diet for 4 wk. It is postulated that the retardation in weight gain of the pups could be due to inadequate milk production.

Inhibition of the hepatocarcinogenicity of aflatoxin B1 in rats by low levels of the phenolic antioxidants butylated hydroxyanisole and butylated hydroxytoluene.

The phenolic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were studied for inhibition of aflatoxin B1 (AFB1) hepatocarcinogenesis in male Fischer 344 rats. The antioxidants were administered at 5, 25, or 125 ppm in AIN-76A diet for 42 weeks. Beginning with week 2, 5 micrograms/kg of AFB1 was given by intragastric instillation three times a week for 40 weeks either alone or concurrently with BHA or BHT feeding. The development of hepatocellular altered foci (HAF) induced by AFB1, as indicators of hepatocarcinogenesis, was monitored using immunohistochemical staining for the placental form of glutathione S-transferase. By 16 weeks the multiplicity of foci was 1.97/cm2 of liver area in rats given only AFB1, and this increased to 4.11/cm2 at 24 weeks and to 10.60/cm2 at 32 weeks. At the final sacrifice at 42 weeks, the multiplicity of foci was 12.90/cm2 compared to 0.75/cm2 in untreated controls. In rats given antioxidants in addition to AFB1, the high dose of BHA reduced the multiplicity to 7.72/cm2 and the high dose of BHT reduced the multiplicity to 9.35/cm2. Lower levels did not reduce foci induction. Thus, in male rats under the conditions of this experiment, the level of 125 ppm of either BHA or BHT inhibited the initiation of hepatocarcinogenesis by AFB1. The BHA effect was slightly greater than that of BHT.

P-241 A study of dynamic contrast-enhanced ultrasonography for detection of small space-occupying lesions of the liver -about usefulness of digital subtraction-

Groups of 40, 29, 39 and 44 F0 rats of each sex were fed a semi-synthetic diet containing butylated hydroxytoluene (BHT) in concentrations to provide intakes of 0, 25, 100 or 500 mg/kg body weight/day, respectively. The F0 rats were mated, and groups of 100, 80, 80 and 100 F1 rats of each sex were formed. After weaning, the highest dose of BHT was lowered to 250 mg/kg/day for the F1 rats. At weaning the BHT-treated F1 rats, especially the males, had lower body weights than the controls and the effect was dose related. The survival of the BHT-treated rats of both sexes was higher than that of the controls. Dose-related increases in the numbers of hepatocellular adenomas and carcinomas were statistically significant in male F1 rats when all groups together were tested for heterogeneity or analysis for trend. The increases in hepatocellular adenomas and carcinomas in treated female F1 rats were only statistically significant for adenomas in the analysis for trend. All hepatocelullar tumours were detected when the F1 rats were more than 2 years old.

Very early changes in pulmonary protein kinase C-α and calpain II contents following injection of butylated hydroxytoluene (BHT) into mice

Butylated hydroxytoluene (BHT)-induced lung damage in mice is an excellent model system for studying mechanisms of chemically-induced, reversible alveolar injury. Changes in the pulmonary contents of protein kinase C (PKC) and the calcium-dependent protease, calpain, were previously noted during the repair phase following BHT-induced pneumotoxicity. Calpain is believed to initiate PKC down-regulation. PKC-α is the major PKC isozyme and calpain II the major calpain isozyme in mouse lung. We have now studied the time course of these enzymatic changes in detail. Pulmonary PKC-α concentrations decreased as early as 45 min after an i.p. injection of 200 mg/kg BHT. Calpain II levels rose within the first 40 min after BHT injection, and then declined below control levels. The rapidity of these changes implies a role of these enzymes in mediating the onset of injury. Lung damage and repair, as estimated by measuring the lung weight/body weight ratio, is maximal 6 days after administration of this dose of BHT. The extent of the decreased PKC-α and calpain II concentrations at this time was linearly related to the estimated degree of injury based on increased lung weight. This correlation suggests the value of monitoring these enzymes as putative early biomarkers of alveolar injury.

The dose-dependent effect of BHT (butylated hydroxytoluene) on vitamin K-dependent blood coagulation in rats

Earlier studies have reported a reduction of vitamin K-dependent blood clotting factor activity and incidence of haemorrhagic death in rats fed butylated hydroxytoluene (BHT); however, the vitamin K status of the animals used in these studies was claimed to be inadequate. The aim of the study reported here was to determine the effect of BHT on vitamin K-dependent clotting factors in vitamin K-sufficient and vitamin K-supplemented rats. Rats given BHT (3000 mg/kg body weight) for up to 21 days, in a diet containing a minimum of 3 ppm vitamin K 3 (six times the recommended requirement), showed decreased vitamin K-dependent blood clotting factor activities, demonstrated by increases in factor-specific clotting time assays. Clotting times were prolonged within 7 days, significantly increased within 14 days ( P < 0.001) and maximally increased 5.5-fold at 21 days ( P < 0.05). Supplementation with a further 250 ppm vitamin K 3 reversed this effect. BHT did not increase prothrombin time (PT), the usual index of clotting. However, in a similar 14-day investigation, a small but significant increase in PT (up to 151%, P < 0.005) was seen within 7 days. Further vitamin K supplementation was incapable of reversing this effect completely. A similar trend was shown by activated partial thromboplastin time. The 1/51 dilution Thrombotest, a more sensitive indicator of vitamin K-dependent clotting factor activity in the rat, was significantly increased (more than four fold, P < 0.01) within 7 days. This increase was fully reversed by further vitamin K supplementation. Prolongation of Thrombotest time was significant at a BHT dose level of 600 mg/kg body weight per day and this could be reversed by further supplementation of only 3.0 ppm vitamin K. However, at dose levels of 125 mg BHT/kg body weight per day or less, no clotting defects were observed. These studies confirm that chronic administration of more than 600 mg BHT/kg/day to rats supplied with recommended amounts of vitamin K can depress clotting factors and precipitate haemorrhagic deaths. When further vitamin K is provided, these deaths could be prevented even though not all clotting abnormalities may be reversed. This study disproves the proposal that BHT-related clotting factor defects are confined to rats of inadequate vitamin K status, but shows that such effects do not occur at dose levels lower than 600 mg/kg/day. The results further indicate that rats receiving a high dose of BHT have a higher vitamin K requirement than would otherwise be considered necessary. However, as BHT produces no clotting defects in these animals receiving an intake 1000 times the acceptable daily intake, such clotting effects are most unlikely to indicate a human safety problem for BHT.

Inhibition by butylated hydroxytoluene and its oxidative metabolites of DMBA-induced mammary tumorigenesis and of mammary DMBA-DNA adduct formation in vivo in the female rat

The phenolic food antioxidant butylated hydroxytoluene (BHT) has been reported to inhibit the initiation stage of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis in the female rat. However, the mechanism for this antitumorigenic effect of BHT is unknown. The present studies were conducted to evaluate the relative effect of the parent chemical BHT and two of its major oxidative metabolites, 2,6-di- tert-butyl-4-hydroxymethylphenol (BHT-BzOH) and 2,6-di- tert-butyl-1,4-benzoquinone (BHT-quinone), on DMBA-induced rat mammary tumorigenesis and on the formation of rat mammary DMBA-DNA adducts in vivo. The ip administration of either BHT or BHT-quinone at 200 mg/kg body weight for 2wk before until 1 wk after DMBA administration inhibited the development of mammary tumours as compared with controls. The extent of tumour inhibition by BHT (39%) was greater than that exhibited by BHT-quinone (25%). The administration of BHT-BzOH at 200 mg/kg body weight did not inhibit mammary tumorigenesis. Thus, the inhibition of DMBA-induced mammary tumorigenesis by BHT does not appear to be mediated by the oxidative BHT metabolites BHT-BzOH or BHT-quinone. In addition, there was a good quantitative correlation between the inhibition of mammary tumorigenesis by BHT and BHT-quinone and their respective abilities to decrease total binding in vivo of DMBA to mammary DNA. The inhibition of specific mammary DMBA-DNA adducts by BHT was not identical to the inhibition of adducts by BHT-quinone. However, the decrease in formation of the major mammary adduct derived from the anti-dihydrodiolepoxide of DMBA bound to deoxyguanosine most closely correlated to the relative abilities of BHT and BHT-quinone to inhibit mammary tumorigenesis. When mammary adduct formation was examined in response to BHT dose, the administration of BHT at doses of 100 mg/kg body weight and 200 mg/kg body weight resulted in the inhibition of anti-derived but not syn-derived mammary DMBA-DNA adducts. Together, these studies suggest that in addition to the inhibition of total mammary DMBA-DNA adduct formation, the inhibition of mammary DNA adducts formed from the anti-dihydrodiolepoxide of DMBA also may be specifically important in the inhibitory effect of BHT on DMBA-induced mammary tumorigenesis.

Haemorrhages due to defective blood coagulation do not occur in mice and guinea-pigs fed butylated hydroxytoluene, but nephrotoxicity is found in mice

Groups of ten male Slc:ddY mice were fed a purified diet containing butylated hydroxytoluene (BHT) at levels of 0, 1.35, 1.75, 2.28, 2.96, 3.85 or 5.00%. They were kept in cages with soft-wood chips as bedding for 30 days. Groups of five Slc:ddY male mice were kept in cages with stainless-steel wire-mesh bottoms (without wood-chip bedding) and fed BHT at 0, 0.5, 1.0 or 2.0% in the diet for 21 days. Male Crj:Hartley guinea-pigs were given a purified ration containing BHT at levels of 0, 0.125 or 0.25% (five animals per group) for 14 days, or at 0, 0.5, 1.0 or 2.0% for 17 days (six animals per group). When BHT was given to mice housed in the mesh-bottomed cages there were one, one and two deaths during the experiment in the 0.5, 1.0 and 2.0% dose groups, respectively. Lung haemorrhages were observed in these dead mice, but in all other mice and guinea-pigs no haemorrhages were found. Indices of prothrombin time and kaolin-activated partial thromboplastin time were significantly decreased by up to 30 and 40%, respectively, in the mice kept on wood-chip bedding, and by up to 40 and 60% in the mice kept in cages with wire bottoms. In guinea-pigs, the prothrombin index was significantly reduced only in the 1.0% BHT group. We conclude that the BHT-induced lung haemorrhages in mice are not caused by a severe reduction in the coagulation process, as they are in rats, and that BHT does not cause bleeding like that observed in rats. However, dose-related toxic nephrosis was found in mice given 1.35-5.0% BHT in the diet. The nephrotoxic ED 50(1 month) was 2.3 g/kg body weight/day. The results suggest that an extremely large dose of BHT can cause renal toxicity in mice.

The site specificity and sensitivity of the rat liver to butylated hydroxytoluene-induced damage

The food additive butylated hydroxytoluene (BHT) is capable of damaging centrilobular or periportal cells in the liver according to the dose and duration of treatment. The effect of two hepatotoxicity potentiating agents on the site specificity of acute cell damage was investigated in Sprague-Dawley rats. A 500 mg/kg oral dose of BHT did not cause overt hepaticnecrosis or alter the cytochrome P450 concentration, but increased ethoxycoumarin- O-deethylation, implying an alteration in the ratio of P450 isoenzymes. Pretreatment with either phenobarbitone (3 × 80 mg/kg, ip) or the glutathione depleting agent buthionine sulfoximine (900 mg/kg, ip) produced liver necrosis in approximately 50% of animals: mainly in centrilobular areas, but with some necrosis in midzonal or periportal areas. Phenobarbitone and BHT did not significantly change the cytochrome P450 concentration, but did alter the ratio of P450 isoenzymes. In phenobarbitone-pretreated rats centrilobular hepatocyte damage was clearly localized in cells with high immunocytochemical staining for the cytochrome P450 IIB subfamily. Buthionine sulfoximine and BHT reduced the cytochrome P450 concentration without reducing ethoxycoumarin- O-deethylase activity, implying a different alteration in the ratio of P450 isoenzymes. These results indicate that phenobarbitone-inducible enzymes are capable of activating high doses of BHT to reactive oxidizing intermediates, which in the absence of adequate glutathione can cause cell death. Enzymes of the P450 IIB subfamily are implicated in this mechanism.

Comparison of lung injury induced in 4 strains of mice by butylated hydroxytoluene

Butylated hydroxytoluene (BHT) is a phenolic antioxidant which induces lung injury in all strains of mice which have been tested, but not in any other species. The mortality of mice treated with BHT is also highly strain-dependent, with LD 50s ranging from 138 to 1739 mg kg . Despite this wide range of toxic doses, the relationship between lung damage and dose has not been well studied. The data presented here demonstrate that BALB/c, ICR and C57BL/6NHsd mice, with LD 50s of 1739, 1243 and 917 respectively, exhibit similar time courses of repair (as assessed by the incorporation of radiolabelled thymidine into DNA) and pulmonary fibrosis (as assessed by lung hydroxyproline content) when given a single 400 mg kg dose of BHT. SSIn mice, with an LD 50 of approximately 350 mg kg , also exhibited a similar time course of repair when given a single dose of 300 mg kg BHT, although fibrosis did not develop in these animals. These data indicate that all strains of mice develop similar levels of lung injury at equivalent doses and that the extent of lung damage produced in mice does not correlate with the lethal dose.

Short-term effects of butylated hydroxytoluene on the Wistar rat liver, urinary bladder and thyroid gland

Long-term feeding of butylated hydroxytoluene (BHT) to rats and mice has been linked to the enhancement of the incidence of liver, urinary bladder and thyroid of the male Wistar rat, feeding the highest tolerated doses of BHT for 30 days does not lead to detectable increases in [ 3H]thymidine labeling. On the other hand, treatment of rats with 0.5% dietary BHT leads to a time-limited increase in liver cell [ 3H]thymidine labeling that subsided to control values within 8 days. This increase in [ 3H]thymidine labeling in the liver is accompanied by an unexpectedly large increase in the mitotic index. These results are discussed in the light of the behavior of certain rodent liver tumorigens.

A metabolite of butylated hydroxytoluene with potent tumor-promoting activity in mouse lung.

Chronically administered butylated hydroxytoluene (BHT) can enhance the formation of carcinogen-induced lung tumors in mice, and biotransformation of BHT is required for this activity. In the present study, oxidative metabolism of BHT by liver microsomes and lung tumor promotion by BHT in three inbred mouse strains, NGP/N, A/J and MA/MyJ, were compared. The strain order shown is the order of increasing susceptibility of these mice to BHT tumor promotion which correlates with their increasing ability to produce a particular metabolite, BHT-BuOH, by hydroxylation of BHT at one of the tert-butyl groups. Chronic BHT administration, according to the treatment regimen for promotion, selectively induced the BHT oxidization pathway leading to BHT-BuOH. The results suggest that formation of BHT-BuOH leads to the tumor promoting effects of BHT. This hypothesis was tested directly by chronic administration of BHT, BHT-BuOH, and two other metabolites, 2,6-di-tert-butyl-4-hydroxymethyl phenol and 2,6-di-tert-butyl-1,4-benzoquinone to MA/MyJ mice following a single injection of urethane. The only metabolite that enhanced lung tumor formation was BHT-BuOH, and it was effective at one-fourth the effective dose of BHT. Thus both indirect and direct evidence implicates BHT-BuOH formation as an important step in the chain of events leading to promotion of lung tumors.

Disposition of single oral doses of butylated hydroxytoluene in man and rat

The kinetics and metabolism of butylated hydroxytoluene (BHT) in man and rats have been compared. Single oral doses of 200, 63 or 20 mg BHT/kg body weight were administered to rats and a single oral dose of 0.5 mg/kg body weight was ingested by human volunteers (non-smoking males). In rats, kinetic parameters (area under the plasma concentration-time curve, plasma BHT peak levels) showed a dose-dependent increase. Plasma BHT levels after oral administration were about four times higher than those that have been reported for another synthetic food antioxidant, butylated hydroxyanisole (BHA; Verhagen et al., Fd Chem. Toxic. 27, 151–158). This may be a reflection of a smaller volume of distribution for BHT, since there were no differences in plasma elimination half-life or plasma clearance between BHT and BHA. In man, the mean plasma concentration-time profile after oral BHT intake was well below the BHT profiles observed for rats and closely followed plasma BHA kinetics in man. In rats, the simultaneous administration of BHT (200 mg/kg body weight) and BHA (200 mg/kg) significantly decreased the absorption of BHT from the gastro-intestinal tract in the first few hours after treatment; the plasma kinetics of BHA were not influenced by the simultaneous administration of BHT. In human female volunteers no alterations in plasma BHT or BHA profiles were seen after the simultaneous ingestion of BHT (0.25 mg/kg body weight) and BHA (0.25 mg/kg). Rats excrete about 10% of an oral dose of 200 mg BHT/kg as unchanged BHT in the faeces, whereas in man no BHT could be detected in the faeces. Urinary excretion of (un)conjugated 3,5-di- tert-butyl-4-hydroxybenzoic acid (BHT-COOH) accounts for only a small percentage of the administered dose in both rats and humans. It is concluded that the plasma BHT concentrations reached after the administration of a single medium to high dose of BHT to rats or a single low dose to man are very different.

2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT quinone methide): an active metabolite of BHT causing haemorrhages in rats.

Male Sprague-Dawley rats and male ICR mice, species respectively susceptible and resistant to the haemorrhagic effect of butylated hydroxytoluene (BHT), were administered BHT quinone methide (2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone) orally; 24 or 48 h later the plasma concentrations of blood coagulation factors II (prothrombin), VII, IX and X were determined. BHT quinone methide caused a decrease in factors II, VII, IX and X in a dose-dependent manner after 48 h, while a similar dose of BHT did not. Haemorrhages in epididymis or thymus were found in BHT quinone methide-treated rats. These findings may support the belief that BHT quinone methide is an active metabolite which disturbs the vitamin K redox cycle in BHT-induced haemorrhage.

Enhancement of butylated hydroxytoluene-induced mouse lung damage by butylated hydroxyanisole

The phenolic antioxidant butylated hydroxytoluene (BHT) is known to produce a dose-dependent increase in mouse lung weight which is characterized by the necrosis of pulmonary type I and endothelial cells. We studied the ability of butylated hydroxyanisole (BHA) to modify BHT-induced changes in lung weight in male CD-1 mice. BHA alone had no effect on lung weight up to a dose of 500 mg/kg (sc). However, when injected 30 minutes prior to subthreshold doses of BHT (0–250 mg/kg, ip), BHA significantly enhanced lung weight in a dose-dependent manner. The ability of BHA to enhance BHT-induced changes in lung weight was dependent on both the time and the route of administration of BHA relative to BHT. Deuteration of BHT abolished the in vivo toxicity from the combination of BHA and BHT. These results suggest that the toxicity resulting from the combination of BHA and BHT is due to the formation of BHT-quinone methide and that the role of BHA might be either to deplete some protective mechanism in the target pulmonary cells or to enhance the biotransformation of BHT into BHT-quinone methide.

Butylated hydroxytoluene (BHT)-mediated increases in NAD(P)H-quinone oxidoreductase (QR) in mouse lung: Evidence for the alveolar type II cell as a site of QR activity

Administration of butylated hydroxytoluene (BHT) to inbred A J mice increased lung cytosolic NAD(P)H-quinone oxidoreductase (QR) activity. Increased QR activity was observed using a dose of BHT of 100 mg/kg ip, although maximal increases in activity were observed following BHT 300 mg/kg ip. Increased lung QR activity could be observed as early as 24 hr post-BHT (300 mg/kg ip). Bronchiolar Clara cells and pulmonary macrophages isolated using an elastase digestion procedure from both control and BHT-treated mice exhibited minimal QR activity. The majority of basal and BHT-increased QR activity remained in the undigested lung. As isolation of many cell types to a high degree of purity from mouse lung is not yet feasible, QR activity was measured in established mouse lung cell lines and in cell isolates from rat lung. QR activity was low in a Clara tumor cell line, but higher activity was observed in three alveolar type II cell lines. Type II cell-enriched populations isolated from rat lung exhibited sixfold greater QR activity than the remaining undigested lung. Pulmonary QR activity was significantly greater in rat than in A J or C57 mice. Treatment of cultured Clara or type II cells with BHT in vitro did not increase QR activity.

Biochemical effects of two promoters of hepatocarcinogenesis in rats

The effects of two promoters of hepatocarcinogenesis-phenobarbital and butylated hydroxytoluene (BHT)—on five hepatic biochemical parameters were examined in adult female rats. Phenobarbital given orally in two doses each of 110 mg/kg 21 and 4 hr before the rats were killed caused large increases in hepatic ornithine decarboxylase (ODC) activity and cytochrome P-450 content. Extending the number of phenobarbital treatments to five increased the hepatic enzyme induction and also caused a minor decrease in hepatic glutathione and a small increase in serum alanine aminotransferase activity. Two oral doses of 700 mg BHT/kg (20% of the LD 50) caused hepatic DNA damage and induction of both ODC activity and cytochrome P-450 content. When the dose of BHT was reduced from 700 to 140 mg/kg no significant effects on the biochemical parameters were found. Both promoters of hepatocarcinogenesis were identified by their induction of ODC, a marker for promotional potential, but only BHT showed a potential for carcinogenic initiation. The biochemical parameters examined, particularly the alkaline elution technique for DNA damage, ornithine decarboxylase activity and serum alanine aminotransferase, may constitute a useful assay system for examining a compound's potential for carcinogenic initiation, carcinogenic promotion and cellular toxicity.

Decrease in blood coagulation factors II (prothrombin), VII, IX and X in the rat after a single oral dose of butylated hydroxytoluene

Male Sprague-Dawley rats were given butylated hydroxytoluene (BHT) in a dose of 800 mg/kg body weight orally, and 0.5–72 hr later plasma concentrations of factors II, VII, IX and X and hepatic levels of BHT and BHT quinone methide (2,6-di- tert-butyl-4-methylene-2,5-cyclohexadienone) were determined. Levels of factors II, VII, X and IX were reduced 36–60 hr after BHT treatment, but by 72 hr, those most affected (VII and IX) showed some recovery and X had returned to normal. Hepatic levels of BHT reached a maximum 3 hr (a major peak) and 24 hr after BHT dosing and BHT quinone methide reached a maximum at 6 and 24 hr (a major peak). In rats given BHT orally in doses of 200, 400 and 800 mg/kg, factors II, VII and X decreased after 48 hr only in rats given the highest dosage, but factor IX was more susceptible to BHT and showed a dose-dependent decrease. Phylloquinone (1 mg/rat) injected ip 24 hr after the administration of 800 mg BHT/kg maintained normal levels of factors VII and X and an almost normal level of factor IX, but had little effect on the level of factor II. In studies of the effects of drug-metabolizing-enzyme modifiers, neither ip pretreatment with 75 mg phenobarbital sodium/kg for 3 days nor the feeding of 1% cysteine in the diet throughout the experiment prevented the decrease in vitamin-K-dependent factors by 800 mg BHT/kg, but 2-day ip pretreatment with 60 mg cobaltous chloride/kg/day maintained normal levels of factors II and VII and reduced the BHT effect on factors IX and X. SKF 525A (50 mg/kg) injected ip either 30 min before or 12 hr after BHT treatment partially prevented the decrease in factors II, VII and X, or in all four factors, respectively. Thus the decrease in vitamin K-dependent factors may be the same with a single oral dose of BHT as with dietary BHT, and the anticoagulant effect may require the metabolic activation of BHT.