The effect of indomethacin, prednisolone and Cis-4-hydroxyproline on pulmonary fibrosis produced by butylated hydroxytoluene and oxygen

The purpose of this study was to examine whether development of pulmonary fibrosis in mice could be influenced by indomethacin, prednisolone or a proline analog. Pulmonary fibrosis was produced in mice treated with butylated hydroxytoluene (BHT) 400 mg/kg and immediately exposed to 80% oxygen for 3 days. This treatment regimen resulted in 47% mortality. Surviving mice exhibited significant accumulations of pulmonary collagen as evidenced by increases in total lung hydroxyproline levels. The administration of indomethacin (4 mg/kg/day) on days 1–6 after BHT decreased mortality to 14% and diminished the accumulation of collagen in lung tissue. Indomethacin also enhanced survival when administered on days 1–3 after BHT/O₂ but had no effect on lung collagen levels. Treatment with indomethacin on days 4–6 after BHT had no beneficial effect. The administration of prednisolone (60 mg/kg/day) on days 1–3, 1–6 or 4–6 after BHT decreased mortality but had no effect on accumulation of lung collagen. Cis-4-hydroxyproline (400 mg/kg/day) also had no effect on pulmonary fibrosis but did enhance survival when given on days 1–3 after BHT. Administering prednisolone (60 mg/kg/day) on days 1–6 after BHT to mice left in room air produced significantly more pulmonary fibrosis than in BHT-treated mice given saline. These data support the use of the BHT/O₂ model of pulmonary fibrosis for screening potential antifibrotic agents. The possibility that corticosteroid treatment may enhance pulmonary fibrosis in a damaged lung is also demonstrated.     

Effect of butylated hydroxytoluene on the lipid composition of rat liver

Hepatic lipids were studied in Sprague-Dawley male rats given butylated hydroxytoluene (BHT) at a level of 1.20% for 1 week. BHT significantly increased cholesterol esters and phospholipids but decreased triglycerides, non-esterified fatty acids and diglycerides. BHT also increased phosphatidylethylanolamine or decreased phosphatidylinositol and lysophosphatidylcholine. Fatty acid composition of each lipid class was also changed by BHT-feeding. The decrease in $\text{16:}\text{1}\text{16}\text{:0, 18:}\text{1}\text{18}\text{:0}\text{and}\text{20:}\text{4}\text{18}\text{:2}$ ratios of total lipids, non-esterified fatty acids or phospholipids of BHT-given rats suggests that BHT decreases the activity of fatty acid desaturase in the liver.     

Long-term studies on chemically induced liver enlargement in the rat I. Sustained induction of microsomal enzymes with absence of liver damage on feeding phenobarbitone or butylated hydroxytoluene

As part of a study on the relationships between liver enlargement, induction of drug-metabolising enzymes and certain forms of liver damage, sequential biochemical and morphological observations were made on the livers of female rats fed diets containing 0.4% butylated hydroxytoluene (BHT) or 0.25% phenobarbitone for periods of up to 80 weeks. Both compounds increased the relative liver weight and produced marked enhancement of the activities of ethylmorphine N-demthylase, aniline 4-hydroxylase, biphenyl 4-hydroxylase and NADPH-cytochrome c reductase and the contents of cytochromes P-450 and b₅ and microsomal protein. These changes were evident after 1 week of treatment and persisted until treatment was stopped after 80 weeks. The only morphological changes observed throughout the period of treatment were centrilobular cell enlargment and hypertrophy of the smooth endoplasmic reticulum (SER). Histochemical examination revealed a centrilobular depression of glucose-6-phosphatase but there was no disturbance of the normal pericanalicular distribution of lysosomes. All changes observed with either compound were reversible on cessation of treatment at week 80.These results support the concept that liver enlargement accompanied by induction of drug-metabolising enzymes represents an adaptive response and they provide a basis for the interpretation of pathological changes developing in the enlarged liver unaccompanied by drug-metabolising enzyme induction.     

Feeding of butylated hydroxytoluene to rats caused a rapid decrease in blood coagulation factors II (prothrombin), VII,IX and X

Male Sprague-Dawley rats were fed a diet containing 1.2% butylated hydroxytoluene (BHT) for 1–7 days, and blood coagulation factors II, VII, VIII, IX and X, and platelet aggregation were measured. The plasma concentrations of factors II, VII, IX and X were significantly reduced in a time-dependent fashion when BHT was administered for 2–7 days and haemorrhages in epididymis were found in rats given BHT for 4–7 days. On the contrary, thrombin-induced and calcium-required aggregation of washed platelets was unchanged throughout the experiment. These results suggest that factors II, VII, IX and X rapidly decrease immediately after the administration of BHT, but hypoaggregability of washed platelets reported previously may be a secondary defect caused by bleeding.This paper was presented at the 58th annual meeting of Japanese Pharmacological Society, March 26–29, 1985, Tokyo, Japan     

Enhancement of allergic responses in vivo and in vitro by butylated hydroxytoluene

The effect of butylated hydroxytoluene (BHT), which is used widely as an antioxidant, on IgE-dependent allergic responses in vivo and in vitro was investigated. For in vivo study, passive cutaneous anaphylaxis (PCA) was elicited in rats by i.d. injection of anti-DNP IgE and 48 h later by i.v. injection of DNP-HSA. BHT was i.p. given immediately after anti-DNP IgE injection. For in vitro studies, the rat mast cell line RBL2H3 sensitized with monoclonal anti-dinitrophenol (DNP) IgE was challenged with the multivalent antigen DNP-human serum albumin (DNP-HSA) in the presence or absence of BHT. beta-Hexosaminidase and histamine released from RBL2H3 cells, as indicators of degranulation of the cells, the concentration of intracellular Ca2+, the level of phosphorylated-Akt, and global tyrosine phosphorylation as indicators of mast cell activation, were measured. The results showed that BHT given to anti-DNP IgE-sensitized rats augmented DNP-specific PCA in a dose-dependent manner. In the presence of BHT, IgE-induced releases of beta-hexosaminidase and histamine from RBL2H3 cells were increased. BHT also further elevated IgE-mediated increased concentrations of intracellular Ca2+ and the levels of phosphorylated-Akt, but did not affect global tyrosine phosphorylation, in RBL2H3 cells. Moreover, the PI3K inhibitor LY294002 inhibited IgE-dependent degranulation and its enhancement by BHT. These findings indicate that BHT may upregulate PCA by enhancing mast cell degranulation associated with enhancements of intracellular Ca2+ concentration and PI3K activation, suggesting that BHT might affect allergic diseases such as allergic rhinitis and asthma.     

Induction of microsomal N-hydroxylation of N-2-fluorenylacetamide in rat liver.

Single or multiple injections of N-2-fluorenyl-acetamide (2-FAA) to Sprague-Dawley rats increased N-hydroxylation of 2-FAA by hepatic microsomes 3- to 12-fold without changing the content of microsomal hemoprotein (cytochrome P-450 or P₁-450) measured either by carbon monoxide difference spectra, by gel electrophoresis of microsomal preparations or by formation of the ethyl isocyanide cytochrome P₁-450 complex. Carbon monoxide inhibited N-hydroxylation of 2-FAA by hepatic microsomes of 2-FAA-treated rats. Inhibition by carbon monoxide indicated that either cytochrome P-450 or P₁-450 is the terminal oxidase in N-hydroxylation by microsomes of 2-FAA-treated rats. Unlike pretreatment of rats with phenobarbital or 3-methylcholanthrene, pretreatment with 2-FAA did not appear to induce the synthesis of microsomal hemoprotein. The activities of NADPH-cytochrome c reductase, NADPH-cytochrome P-450 reductase and of amine oxidase in microsomes of 2-FAA-treated rats were not increased and thus did not account for the stimulation of N-hydroxylation. The induction, by 2-FAA, of a heretofore unknown electron carrier associated with the hepatic mixed-function oxidase is postulated and under investigation.     

Effect of butylated hydroxytoluene,curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes

1.?The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes.

2.?Treatment of human hepatocytes for 72 h with 2–200 µM TB produced concentration-dependent increases in CYP1A2, CYP2B6 and CYP3A4 mRNA levels, whereas treatment with BHT increased CYP2B6 and CYP3A4 mRNA levels. CYP1A2, CYP2B6 and CYP3A4 mRNA levels were induced around 48-, 21- and 9-fold, respectively, by 200 µM TB, with CYP2B6 and CYP 3A4 mRNA levels being induced around 12- and 7-fold, respectively, by 200 µM BHT.

3.?In contrast, the treatment of human hepatocytes for 72 h with PG and CC had little or no effect on CYP mRNA levels.

4.?The treatment of human hepatocytes with TB also induced CYP1A-dependent 7-ethoxyresorufin O-deethylase activity, whereas BHT induced CYP3A-dependent testosterone 6β-hydroxylase activity.

5.?In summary, the results demonstrate that TB is a mixed inducer of CYP forms in human hepatocytes inducing CYP1A, CYP2B and CYP3A forms, whereas BHT is an inducer of CYP2B and CYP3A forms.     

Antioxidant effect of butylated hydroxytoluene on ferric nitrilotriacetate induced renal hyper proliferation and injury in rats

This study was designed to investigate the effect of butylated hydroxy toluene (BHT), a phenolic antioxidant used in foods, cosmetics and pharmaceutical products, on Fe-NTA-induced nephrotoxicity in rats. Fe-NTA (alone) treatment enhances ornithine decarboxylase activity to 5.3-fold, and [³H] thymidine incorporation in DNA to 3.5-fold compared with the corresponding saline treated control. The enhanced ornithine decarboxylase activity and DNA synthesis showed a reduction to 2.12–2.15-fold respectively at a higher dose of 2 mg BHT/day/animal, compared with the Fe-NTA treated group. Fe-NTA treatment also enhanced the renal microsomal lipid peroxidation to 2.0-fold and decreased the activities of glutathione and antioxidant enzymes to a range of 2.2–2.5-fold in kidney. These changes were reversed significantly in animals receiving a pretreatment of BHT. Present data suggests that BHT can prevent the toxic effects of Fe-NTA and can serve as a potent chemopreventive agent to suppress oxidant-induced tissue injury and nephrotoxicity in rats.     

Long-term studies on chemically induced liver enlargement in the rat I. Transient induction of microsomal enzymes leading to liver damage and nodular hyperplasia produced by safrole and ponceau MX

Sequential biochemical and morphological observations were made on the livers of female rats fed a diet containing 0.25% safrole or 1.0% Ponceau MX for up to 85 weeks. After administration of safrole for 7 days the relative was markedly enhanced. This enzyme induction was short-lived, however, and continued administration of safrole led to inhibition of drug-metabolising activity. Despite this the liver remained enlarged and concentrations of NADPH-cytochrome c reductase, cytochrome b₅ and microsomal protein were markedly elevated. During the initial phase of enzyme induction no histochemical or morpholigical abnormalities were evident but the loss of this inductive response was associated with histochemical and ultrastructural evidence of liver damage. The enlarged liver produced by Ponceau MX was initially accompanied by moderate induction of the drug-metabolising enzymes followed by gradual reversal as treatment continued. Histochemical evidence of liver damage was encountered after only 1 week of treatment. The early cytological changes produced by both compounds were followed by the development of fatty change, cell necrosis and, ultimately, hepatic nodules.The relationship between an absence of sustained enzyme induction and the development of pathological changes in the enlarged liver is discussed     

The effects of butylated hydroxytoluene on the in vitro metabolism, DNA-binding and mutagenicity of aflatoxin B1 in the rat

Butylated hydroxytoluene pretreatment in the rat enhanced the total in vitro metabolism of aflatoxin B1 by the hepatic postmitochondrial fraction (S-9) and increased the formation of aflatoxin M1, aflatoxin Q1 and a metabolite tentatively identified as the aflatoxin-glutathione conjugate, the latter being the major metabolite produced. Addition of diethyl maleate, a glutathione depletor, to the incubation mix, reduced formation of the conjugate. No significant difference between treated and control animals was observed in the S-9-mediated binding of aflatoxin B1 to calf thymus DNA. However, the mutagenicity of aflatoxin B1 in Salmonella typhimurium TA98 was significantly lower in the presence of S-9 from BHT-treated rats than with S-9 from controls.     

An enhancing effect of the antihistaminic drug methapyrilene on rat liver carcinogenesis by previously administered N-2-fluorenylacetamide

The effect on hepatocarcinogenesis of sequential administration of the antihistaminic drug methapyrilene (MP) or the typical liver tumor promoter, phenobarbital (PB), each given after the genotoxic liver carcinogen, N-2-fluorenylacetamide (FAA) was studied. An initial exposure to 0.02% of FAA in the diet for 8 weeks was used to produce hepatocellular altered foci. Rats maintained for an additional 24 weeks on basal diet developed a low incidence of liver neoplasms. MP at 500 and 1000 ppm in the diet for 24 weeks after FAA increased the frequency of altered foci at early stages and liver neoplasms later, as did PB. Neither MP nor PB alone produced neoplasms, but MP alone produced a significant incidence of altered foci. Therefore, the results provide evidence for a promoting action of MP, but additional effects may be involved in its carcinogenicity.     

Covalent binding of benzo[a]pyrene to rat liver cytosolic proteins and its effect on the binding to microsomal proteins

Benzo[a]pyrene will bind covalently to rat liver cytosolic proteins when incubated with microsomes and NADPH. The binding is most extensive when microsomes from 3-methylcholanthrene-treated rather than phenobarbital-treated or control rats are used. The binding to cytosolic proteins increases when incubations are performed with increasing concentrations of cytosol. At the same time the covalent binding of benzo[a]pyrene to microsomal proteins decreases. Two cytosolic polypeptides are the main targets for benzo[a]pyrene. These have the same mobility in polyacrylamide gels as the subunits of purified glutathione S-transferase B. These subunits also react covalently with benzo[a]pyrene when the transferase is incubated with microsomes and NADPH.     

Effect of butylated hydroxytoluene pretreatment on the excretion, tissue distribution and DNA binding of [14C]aflatoxin B1 in the rat

The effect of butylated hydroxytoluene (BHT) pretreatment (0.5% in the diet for 10 days) on the excretion, tissue distribution and DNA binding of orally administered [14C]aflatoxin B1 (AFB1) was determined in male Fischer F344 rats. The amount of radioactivity excreted in the urine and faeces by 24 hr was higher in BHT-treated rats than in controls. Treatment with BHT enhanced the excretion of water-soluble metabolites in the urine and in the large intestines plus faeces at the earlier sampling times. The amount of radioactivity bound to hepatic nuclear DNA was six times less in the BHT-pretreated rats than in controls 6 hr after administration of the isotope. The half-lives of [14C]DNA in the rat liver were 30 and 46 hr for control and BHT-pretreated rats, respectively. These results indicate that BHT pretreatment may protect the animal from the carcinogenic effects of AFB1 by enhancing the detoxification and excretion of the mycotoxin.     

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.     

Absence of a promoting or sequential syncarcinogenic effect in rat liver by the carcinogenic hypolipidemic drug nafenopin given after N-2-fluorenylacetamide

The hypolipidemic agent nafenopin, (NF), has been reported to be carcinogenic to rat liver. To determine whether nafenopin exerts a promoting or syncarcinogenic effect in rat liver, its effect on liver carcinogenesis induced by N-2-fluorenylacetamide (FAA) was studied. In two separate experiments, male F344 rats were fed 0.02% FAA for either 10 or 8 weeks to induce preneoplastic liver lesions. Following a recovery period of 1 week, rats were given 0.01 or 0.02% NF in the diet for 23 weeks in one experiment and 0.05 or 0.1% for 24 weeks in the other. The final incidence of neoplasms, and their numbers, size distribution, and degrees of differentiation were not significantly different in groups given NF after FAA compared to those maintained on a basal diet after FAA. In the group treated with the highest dose level of NF following FAA, however, there was a decrease in the number of grossly visible small neoplasms. In contrast, the liver neoplasm promoter phenobarbital increased the multiplicity, although not the incidence, of liver neoplasms when given after FAA. Thus, four different dose levels of NF showed no promoting or syncarcinogenic effect on FAA-induced hepatocarcinogenesis.     

Selective induction of apoptosis in mouse and human lung epithelial cell lines by the tert-butyl hydroxylated metabolite of butylated hydroxytoluene: a proposed role in tumor promotion

Butylated hydroxytoluene (BHT) causes lung injury in mice and promotes tumor formation. Hydroxylation of a tert-butyl group on BHT to yield the metabolite, 6-tert-butyl-2-[2′-(2′-hydroxymethyl)-propyl]-4-methylphenol (BHTOH), may be required. BHTOH is more potent than BHT on an equimolar basis in causing lung damage, enhancing lung tumor development, killing isolated bronchiolar non-ciliated Clara cells, and inhibiting lung epithelial gap junctional intercellular communication. One mechanism proposed for tumor promoting agents is selective cytotoxicity; killing normal cells allows uninhibited clonal expansion of neighboring initiated cells. We compared the abilities of BHT, BHTOH, and other BHT metabolites to kill non-tumorigenic and tumorigenic mouse and human lung cell lines, and examined the contribution of apoptosis to this cytotoxicity. These cells lack the cytochrome P450 2B isozyme necessary for converting BHT to BHTOH. BHTOH and 4-hydroperoxy-4-methyl-2,6-di-tert-butyl-2,5-cyclohex-adienone (BHTOOH) were most toxic, BHT and 2,6-di-tert-butyl-1,4-benzoquinone (BHTQu) were less potent, and 4-methyl BHT metabolites that are not pneumotoxic were ineffective. BHTOH most strongly induced apoptosis, based on nuclear condensation and transmission electron microscopy. Non-tumorigenic cells were as susceptible to cell death as the neoplastic cell lines when apoptosis and necrosis are not distinguished, but more sensitive to BHTOH-induced apoptosis. An apoptotic mechanism may underlie the lung tumor promoting actions of BHTOH.     

Microsomal metabolism of arylamides by the rat and guinea pig—I.: Oxidation of N-3-fluorenylacetamide at carbon-atom-9—A major metabolic reaction

The N-hydroxylation of N-3-fluorenylacetamide (3-FAA), an isomer of the carcinogen, N-2-fluorenylacetamide (2-FAA), by hepatic microsomes of untreated and 3-methylcholanthrene (3-MC)-treated guinea pigs was found to be of a similar low order as that previously observed in the rat. Hepatic microsomes of the guinea pig and of the rat converted 3-FAA to N-(9-hydroxy)-3-FAA and to N-(9-oxo)-3-FAA. These new metabolites were separated and identified by high-pressure liquid chromatography (h.p.l.c.). N-(9-hydroxy)-3-FAA was the major product of the hydroxylation of 3-FAA by hepatic microsomes of the rat or guinea pig exceeding the formation of N-(7-hydroxy)-3-FAA, the principal phenolic metabolite of 3-FAA. The amounts of N-(9-oxo)-3-FAA formed were about one-third of the amounts of N-(9-hydroxy)-3-FAA produced. In contrast to the formation of phenolic metabolites, the hydroxylation of 3-FAA to N-(9-hydroxy)-3-FAA was not increased by pretreatment of guinea pigs or rats with 3-MC. Similarly, pretreatment of rats with PB did not enhance the yield of N-(9-hydroxy)-3-FAA. Co inhibited the formation of N-(9-hydroxy)-3-FAA by 80 per cent. These data lead us to conclude that the formation of N-(9-hydroxy)-3-FAA is catalyzed by a microsomal hemoprotein not identical with cytochrome P₁-450 or P-450. In contrast to 3-FAA, 2-FAA appeared to be a poor substrate for hydroxylation to the N-(9-hydroxy)-2-FAA. The susceptibility of 3-FAA to hydroxylation at carbon-atom 9 of the fluorene moiety may be rationalized by resonance structures in which carbon-atom 9 is positively charged and acts as a electrophilic center. Similar resonance structures cannot be written for 2-FAA.     

Protective effect of diphenyl diselenide on acute liver damage induced by 2-nitropropane in rats

The effect of diphenyl diselenide, (PhSe)2, administration on 2-nitropropane (2-NP)-induced hepatic damage was examined in male rats. Rats were pre-treated with a single dose of diphenyl diselenide (10, 50 or 100 micromol/kg). Afterward, they received only one dose of 2-NP (100 mg/kg body weight dissolved in olive oil). The parameters that indicate tissue damage such as plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alpha-fetoprotein (AFP), creatinine and urea were determined. Since toxicity induced by 2-NP is related to oxidative stress, lipid peroxidation was also evaluated. Diphenyl diselenide (100 micromol/kg) significantly reduced plasma ALT, gamma-GGT, AFP levels when compared to 2-NP group. Treatment with diphenyl diselenide, at all doses, effectively protects the increase of lipid peroxidation when compared to 2-NP group. Histological examination revealed that 2-NP treatment causes a moderate swelling and degenerative alterations on hepatocytes and diphenyl diselenide (100 micromol/kg) protects against these alterations. Diphenyl diselenide (50 and 100 micromol/kg) significantly decreased the urea level. This study evidences the protective effect of diphenyl diselenide by 2-NP-induced acute hepatic damage.