Mouse hepatic glutathione transferase isoenzymes and their differential induction by anticarcinogens. Specificities of butylated hydroxyanisole and bisethylxanthogen as inducers of glutathione transferases in male and female CD-1 mice.

GSH transferase isoenzymes of class Mu (two forms), class Pi (one form) and class Alpha (two forms) were purified from liver cytosols of female CD-1 mice pretreated with an anticarcinogenic inducer, 2(3)-t-butyl-4-hydroxyanisole. GSH transferases GT-8.7, GT-8.8a and GT-8.8b, GT-9.0, GT-9.3, GT-10.3 and GT-10.6 contained a minimum of six types of subunits distinguishable by structural, catalytic and immunological characteristics. H.p.l.c. analysis of the subunit compositions of affinity-purified GSH transferases from liver cytosols of induced and non-induced male and female CD-1 mice showed that two anticarcinogenic compounds, 2(3)-t-butyl-4-hydroxyanisole and bisethylxanthogen, differed markedly in their specificities as inducers of GSH transferase.     

Inhibition of the mono-oxygenase system by butylated hydroxyanisole and butylated hydroxytoluene

The commonly used food-additive antioxidants, butylated hydroxyanisole and butylated hydroxytoluene, are inhibitors of the hepatic microsomal mono-oxygenase system, as assayed by benzpyrene hydroxylase activity and demethylase activities. Generally, butylated hydroxyanisole is a more potent inhibitor than butylated hydroxytoluene. Both inhibitors bind to cytochrome P-450 and induce “type I” binding spectra. Cytochrome P-450 is tentatively assigned as the site of inhibition.     

Cytotoxicity of butylated hydroxyanisole in Vero cells

Butylated hydroxyanisole (BHA) is perhaps the most extensively used synthetic antioxidant in the food and cosmetic industry, although considerable controversy exists in the literature regarding the safety of this compound. Most in vitro studies describing the effects of BHA have been performed in cancer cells, but it is unclear whether normal cells are equally susceptible to BHA exposure. The present study investigate the toxic potential of BHA in mammalian cells, using biochemical and morphological parameters, which reveal interference with structures essential for cell survival, proliferation and/or function. Cell growth inhibition was assessed by using colorimetric assays, whereas cellular alterations after BHA exposure, were evaluated using conventional light and fluorescence microscopy. Low doses of BHA exerted a significant cytotoxic effect, associated with loss of mitochondrial function. As the concentration of BHA was increased, morphological alterations in critical subcellular targets such as lysosomes, mitochondria and actin cytoskeleton, were observed. In parallel, BHA induced an irreversible loss of cell proliferative capacity, preceding apoptosis induction. Thus, the dose-dependent activity of BHA on Vero cells appears to be cytotoxic as well as cytostatic. Our observations, although simplified with respect to the in vivo situations, allowed the assessment of the specific damage at the cellular level, and provide some clue about the effects of BHA in non-tumoral mammalian cells.     

Comparative effect of the induction of the subunits of rat liver glutathione S-transferases by butylated hydroxytoluene

When butylated hydroxytoluene (BHT) was administered to rats, the smallest subunit Ya (Mr 22,000) of rat liver GSH S-transferases was found to undergo maximum induction. It is suggested that the differential induction of GSH S-transferase activities by BHT towards different substrates may be due to the differences in the induction of the constituent subunits of GSH S-transferases.     

Tissue-specific expression and subcellular distribution of murine glutathione S-transferase class kappa.

Class kappa glutathione S-transferases are a poorly characterized family of detoxication enzymes whose localization has not been defined. In this study we investigated the tissue, cellular, and subcellular distribution of mouse glutathione S-transferase class kappa 1 (mGSTK1) protein using a variety of immunolocalization techniques. Western blotting analysis of mouse tissue homogenates demonstrated that mGSTK1 is expressed at relatively high levels in liver and stomach. Moderate expression was observed in kidney, heart, large intestine, testis, and lung, whereas sparse or essentially no mGSTK1 protein was detected in small intestine, brain, spleen, and skeletal muscle. Immunohistochemical (IHC) analysis for mGSTK1 revealed granular staining of hepatocytes throughout the liver, consistent with organelle staining. IHC analysis of murine kidney localized GSTK1 to the straight portion of the proximal convoluted tubule (pars recta). Staining was consistent with regions rich in mitochondria. Electron microscopy, using indirect immunocolloidal gold staining, clearly showed that mGSTK1 was localized in mitochondria in both mouse liver and kidney. These results are consistent with a role for mGST K1-1 in detoxification, and the confirmation of the intramitochondrial localization of this enzyme implies a unique role for GST class kappa as an antioxidant enzyme.     

Inhibition of murine malaria (Plasmodium chabaudi) in vivo by recombinant interferon-gamma or tumor necrosis factor, and its enhancement by butylated hydroxyanisole

Cell-mediated immunity to malaria may involve macrophages, the monokines that mediate endotoxicity, and reactive oxygen species. Since interferon-gamma activates macrophages to release reactive oxygen species, and tumor necrosis factor-alpha (TNF-alpha) helps both to mediate endotoxicity and to induce leukocytes to secrete reactive oxygen, we monitored the effects of administering recombinant forms of these cytokines on Plasmodium chabaudi adami infections in mice. We also fed infected mice a diet containing 0.75% butylated hydroxyanisole, a scavenger of free radicals. Infections were suppressed by daily i.p. injections of 5 x 10(4) U of recombinant mouse interferon-gamma from day -1 or by recombinant human TNF released from i.p. osmotic pumps at the rate of 6 x 10(3) U/hr. Degenerate intraerythrocytic parasites (crisis forms) were evident much sooner in the course of the suppressed infections, and parasitemias fell correspondingly earlier. The butylated hydroxyanisole diet, in contrast, enhanced the infections. In these mice crisis forms were seen later, and at higher parasitemias, than they normally occur. These observations are consistent with the concept that T cell-dependent, macrophage-derived mediators are central to the type of malarial immunity that kills parasites inside circulating red cells. They also suggest, but do not prove, that both TNF and reactive oxygen species are involved, and that the role of TNF may be more indirect, although no less important, than that of reactive forms of oxygen.     

Tissue-specific regulation of two functional malic enzyme mRNAs by triiodothyronine

Rat liver malic enzyme (ME) synthesis is known to be regulated by 3,5,3'-triiodo-L-thyronine (T3). Hybridization of 32P-labeled ME cDNA with RNA extracted from normal and T3-induced livers (15 or 50 micrograms/100 g body weight for 10 days) showed an increase in the ME mRNA concentration by approximately 11-fold in T3-treated rats. ME activity and ME mass were stimulated to the same degree as ME mRNA. Northern blot analysis of either total or poly(A+) RNA revealed two distinct ME mRNAs (21 and 27 S) which were equally induced by T3 treatment. Both mRNAs were shown by in vitro translation assay to program the synthesis of the same immunoprecipitable protein corresponding to full-sized ME. From all the above, we concluded that both messages code for active enzyme. ME activity and ME mRNA were also detected in nonhepatic tissues for which different responses to T3 induction were observed without direct correlation with their respective content of T3 nuclear receptor. Increases in ME activity and level of hybridizable ME mRNA were seen 48 h after a single administration of T3 (200 micrograms/100 g body weight) in liver, kidney, and heart (10.3- and 15.5-, 1.7- and 2.6-, and 1.72- and 3.4-fold above basal values, respectively). Lower levels of induction could already be detected after 24 h, liver being the most stimulated tissue. ME was not affected in brain, lung, testis, and spleen. Northern blot analysis showed that both ME mRNAs are present in all tissues tested, although in different relative proportions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrinsic fluorescence quenching of glutathione transferase pi by glutathione binding.

The binding of the GSH to the GSH transferase pi quenches the protein intrinsic fluorescence more than the binding of GS-Me. The calculated dissociation constants are 38.6 microM and 90.9 microM for GSH and GS-Me, respectively. From the reported data it is evident that the binding of GSH to GSH transferase pi quenches the intrinsic fluorescence with two different mechanisms. The first one is a conformational change induced by the binding of the GSH and it is present also with the GS-Me binding. A second proposed mechanism is a contact quenching between the sulphydryl GSH group and a tryptophan residue. This suggests that at least one of the tryptophan residues is located near the GSH binding site.     

The glutathione conjugates of tert-butyl hydroquinone as potent redox cycling agents and possible reactive agents underlying the toxicity of butylated hydroxyanisole.

The glutathione conjugates of tert-butyl hydroquinone, a metabolite of butylated hydroxyanisole (BHA), possess redox potentials which are much higher as compared to the non-conjugated hydroquinone (0.36 V for the hydroquinone and 1.2-1.4 V for the conjugates). As a result, the redox cycling activity of the conjugates, as measured by oxygen consumption in the presence of a reducing agent, is increased tenfold as compared to the non-conjugated hydroquinone. Since evidence for both oxidative damage and the involvement for glutathione in the toxicity of butylated hydroxyanisole is available, this mechanism may be involved in the toxic action of this compound.     

Tissue-specific functions of individual glutathione peroxidases

The family of glutathione peroxidases comprises four distinct mammalian selenoproteins. The classical enzyme (cGPx) is ubiquitously distributed. According to animal, cell culture and inverse genetic studies, its primary function is to counteract oxidative attack. It is dispensible in unstressed animals, and accordingly ranks low in the hierarchy of glutathione peroxidases. The gastrointestinal isoenzyme (GI-GPx) is most related to cGPx and is exclusively expressed in the gastrointestinal tract. It might provide a barrier against hydroperoxides derived from the diet or from metabolism of ingested xenobiotics. The extreme stability in selenium deficiency ranks this glutathione peroxidase highest in the hierarchy of selenoproteins and points to a more vital function than that of cGPx. Plasma GPx (pGPx) behaves similar to cGPx in selenium deficiency. It is directed to extracellular compartments and is expressed in various tissues in contact with body fluids, e.g., kidney, ciliary body, and maternal/fetal interfaces. It has to be rated as an efficient extracellular antioxidant device, though with low capacity because of the limited extracellular content of potential thiol substrates. Phospholipid hydroperoxide glutathione peroxidase (PHGPx), originally presumed to be a universal antioxidant enzyme protecting membrane lipids, appears to have adopted a variety of specific roles like silencing lipoxygenases and becoming an enzymatically inactive structural component of the mitochondrial capsule during sperm maturation. Thus, all individual isoenzymes are efficient peroxidases in principle, but beyond their mere antioxidant potential may exert cell- and tissue-specific roles in metabolic regulation, as is evident for PHGPx and may be expected for others.     

Effects of butylated hydroxyanisole on the aryl hydrocarbon hydroxylase of rats and mice

The effects of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the aryl hydrocarbon hydroxylase (AHH) activities in the liver, lung and skin of rats and mice have been studied to examine the possible mechanisms of the anticarcinogenic actions of these compounds. Both compounds inhibit the hydroxylase activities of hepatic microsomes and nuclei, with BHA a more potent inhibitor than BHT. The AHH of lung microsomes is inhibited to a lesser extent by BHA and BHT than that of the liver. The AHH activities of both liver and lung microsomes become less susceptible to the inhibition after pretreatment of the animals with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but phenobarbital (PB) pretreatment does not produce such an effect. In skin homogenates, however, the AHH activities of control rats and mice are not inhibited by BHA and BHT. The only skin sample which is inhibited by BHA and BHT is that from TCDD-pretreated mice. It has been established that the extent of inhibition with different samples is related to the concentration of BHA in the incubation but not to the amounts or specific activities of microsomes used. Double reciprocal plots suggest that BHA exerts a mixed inhibition on the hydroxylase of liver microsomes with a K_i of 7.7 μM. Analysis of the metabolites of benzo[a]pyrene (BP) shows that BHA inhibits the formation of various metabolites uniformly without changing the regio-selectivity of the enzyme system. The mechanism of inhibition has also been studied with a reconstituted AHH system consisting of cytochrome P-450 (P-450), reductase and phospholipid. The system with P-450 isolated from PB-induced microsomes is inhibited to a much greater extent than that with MC-induced P-450. The results indicate that the inhibitory action of BHA is dependent on the species of the animal, tissue types and treatment with inducers.     

Effects of atrazine, endosulfan and butylated hydroxyanisole on glutathione-S-transferases in Orthosia gothica

Atrazine (1,000 ppm), endosulfan (1 ppm) or butylated hydroxyanisole (BHA) (1,000 ppm) added to a semi-synthetic diet of Orthosia gothica for 2 days in the last instar did not change the soft tissue cytosolic glutathione-S-transferase (GST) activities towards 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and cumene hydroperoxide (CU). However, all three pesticides changed the GST subunit composition compared with the control as observed by reverse phase high performance liquid chromatography of the isozymes purified by glutathione-Sepharose affinity chromatography. The changes seem to have occurred mainly in the GST class 2 subunit. There is no obvious explanation for the changes, which may be a result of interactions between xenobiotic and GST in the cytosol as well as changes in the level of regulation of synthesis. However, the observation added to our knowledge of the processes involved when pesticides are degraded by GSTs in vivo.     

Effect of butylated hydroxyanisole on the toxicity of 2-hydroxy-1,4-naphthoquinone to rats

It has previously been shown that rats pre-treated with butylated hydroxyanisole (BHA), a well-known inducer of the enzyme DT-diaphorase, are protected against the harmful effects of 2-methyl-1,4-naphthoquinone. This is consistent with a role for diaphorase in the detoxification of this quinone, but it is not known if increased tissue levels of this enzyme give protection against other naphthoquinone derivatives. In the present study, rats were dosed with BHA and then challenged with a toxic dose of 2-hydroxy-1,4-naphthoquinone, a substance that causes haemolytic anaemia and renal damage in vivo. Pre-treatment with BHA had no effect upon the nephrotoxicity of 2-hydroxy-1,4-naphthoquinone, but the severity of the haemolysis induced by this compound was increased in the animals given BHA. DT-Diaphorase is known to promote the redox cycling of 2-hydroxy-1,4-naphthoquinone in vitro, with concomitant formation of 'active oxygen' species. The results of the present experiment suggest that activation of 2-hydroxy-1,4-naphthoquinone by DT-diaphorase may also occur in vivo and show that increased tissue levels of DT-diaphorase are not always associated with naphthoquinone detoxification.     

Suppression of glutathione transferase P expression by glucocorticoid.

A strong enhancer element, GPEI, of the glutathione transferase P gene (GST-P) gene is composed of two phorbol 12-O-tetradecanoate 13-acetate (TPA) responsive element (TRE)-like sequences at opposite orientation. Unlike TRE sequences of other genes, GPEI exhibits a strong enhancer activity in F9 cells, which contains little AP-1. GPEI bound to AP-1 In vitro and GST-P expression was activated by TPA and exogenously introduced c-jun gene in a rat fibroblast cell line. Both the stimulated expression of GST-P gene by TPA and that by over-expressed c-Jun were suppressed to the basal level by dexamethasone, an inhibitor of AP-1. Basal expression of GST-P gene, however, was not inhibited by dexamethasone. Transfected chloramphenicol acetyltransferase (CAT) gene having GPEI also behaved as the endogenous GST-P gene. These results indicate that the GPEI is activated by AP-1 but constitutive activity of this enhancer in a rat fibroblast cell line 3Y1 cells is due to some unknown mechanism other than AP-1.