Mkp-1 protects mice against toxin-induced liver damage by promoting the Nrf2 cytoprotective response.

The present study was undertaken to investigate the possible protective effect of mitogen-activated protein kinase phosphatase 1 (Mkp-1) on toxin-induced hepatic injury. Here, we uncovered a positive feedback loop between Mkp-1, a dual threonine/tyrosine phosphatase, and nuclear factor erythroid 2-related factor 2 (Nrf2), a crucial regulator of the defense system in the liver. Mkp-1-/- mice exhibited decreased protein levels of Nrf2, phase II gene products, and reduced glutathione (GSH) in the liver. Induction of detoxifying enzymes by the Nrf2 activator butylated hydroxyanisole (BHA) or sulforaphane, was attenuated in the liver and small intestines of Mkp-1-/- mice, indicating that the Nrf2 signaling pathway is impaired as a result of Mkp-1 deficiency. Mkp-1-/- mice suffered more severe liver injury after a single exposure to hepatotoxin carbon tetrachloride (CCl4) than their wild-type (WT) counterparts. BHA partially rescued the CCl4-induced liver damage in WT mice, but not in Mkp-1-/- mice, suggesting the requirement of Mkp-1 in the activation of Nrf2 signaling against the liver injury. Mechanistically, Mkp-1 upregulated Nrf2 through a direct interaction with the Neh2 domain in the transcription factor, while Nrf2 enhanced the expression of Mkp-1 mRNA by binding to the ARE site at -1719 to -1710bp in the Mkp-1 promoter. Our results reveal novel role of Mkp-1 in the maintenance of redox homeostasis in the liver. Thus, strategies aimed at augmenting Mkp-1 expression may be beneficial in protecting the liver and may provide novel therapeutic approaches to toxin-induced liver injury.

Butylated hydroxyanisole alters rat 5α-reductase and 3α-hydroxysteroid dehydrogenase: Implications for influences of neurosteroidogenesis.

Butylated hydroxyanisole is a synthetic antioxidant. It may affect the function of the nerve system. The objective of the present study is to investigate the direct effects of butylated hydroxyanisole on rat brain neurosteroidogenic 5α-reductase 1 (SRD5A1), 3α-hydroxysteroid dehydrogenase (AKR1C14), and retinol dehydrogenase 2 (RDH2). Rat SRD5A1, AKR1C14, and RDH2 were cloned and expressed in COS1 cells, and the effects of butylated hydroxyanisole on these enzyme activities were measured. Butylated hydroxyanisole inhibited SRD5A1, AKR1C14, and RDH2 with IC50 values of 4.731±0.079µM, 5.753±0.073µM, and over 100µM, respectively. Butylated hydroxyanisole is a competitive inhibitor for both SRD5A1 and AKR1C14. Docking analysis shows that butylated hydroxyanisole binds to the dihydrotestosterone-binding site of AKR1C14. In conclusion, butylated hydroxyanisole is a potent inhibitor of SRD5A1 and AKR1C14, thus reducing the formation of active neurosteroids.

Adverse effect of sub-chronic exposure to benzo(a)pyrene and protective effect of butylated hydroxyanisole on learning and memory ability in male Sprague-Dawley rat.

Previous studies demonstrate that benzo(a)pyrene (B(a)P) can affect hippocampal function and cause spatial cognition impairment. However, the mechanism is incomplete. Some evidence implies that B(a)P may cause an oxidative damage linking to the function of the hippocampus and antioxidant can prevent the oxidative damage in rats, but the ATPase and Ca(2+) in the hippocampus and the protective effect of butylated hydroxyanisole (BHA) have not been studied. This study aimed to investigate the damage of toxicity further induced by B(a)P in hippocampus and the protective effect of BHA. Ninety-six male Sprague-Dawley (SD) rats were randomly divided into four groups (solvent control group, BHA-group, B(a)P-exposed group and B(a)P-BHA-combination group), with daily administration for 90 days. Morris water maze (MWM) was employed to evaluate the learning and memory ability. The levels of malonaldehyde (MDA) content, superoxide dismutase (SOD) activity, Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)ATPase activity in hippocampus were measured by commercial kits. The concentration of Ca(2+) in rat hippocampus was detected by fluorescent labeling. In behavior test it showed that there was an adverse effect on rats in the B(a)P -group. The levels of MDA content and Ca(2+) content were significantly increased in the B(a)P group, while the activities of SOD and ATPase were significantly decreased. In the B(a)P-BHA group, the change of each index diminished significantly. The results suggested that the neurobehavioral toxicity of B(a)P might have a close relationship with oxidative damage, resulted in decreasing of ATPase activities and increasing of Ca(2+) concentration in the hippocampus. Furthermore, BHA can prevent these damages.

Antinociceptive and antioxidant potential of the crude ethanol extract of the leaves of Ageratum conyzoides grown in Bangladesh.

CONTEXT: Ageratum conyzoides Linn. (Asteraceae) is an annual herbaceous plant with a long history of traditional medicinal and agricultural uses; it is usually grown in the northeast part of Bangladesh. OBJECTIVE: The ethanol extract of the plant leaves was evaluated for preliminary phytochemical screening with its antinociceptive and antioxidant activities. MATERIALS AND METHODS: The preliminary phytochemical analysis was performed on the basis of standard procedures. The analgesic activity of the extract was investigated using the acetic acid-induced writhing method in mice. Five complementary tests such as DPPH free radical scavenging, nitric oxide (NO) scavenging, reducing power, Fe(++) ion chelating ability and total phenolic content were used for determining antioxidant activities. RESULTS: The results of preliminary phytochemical analysis showed the presence of alkaloids, reducing sugars, saponins, gums, steroids, tannins and flavonoids. The extract possessed a significant dose-dependent DPPH free radical scavenging activity with an IC50 value of 18.91 µg/ml compared to ascorbic acid (IC50: 2.937 µg/ml) and butylated hydroxyanisole (IC50: 5.10 µg/ml). The IC50 value of the extract for NO scavenging (41.81 µg/ml) was also found to be significant compared to the IC50 value of ascorbic acid (37.93 µg/ml). Moreover, the extract showed reducing power activity and Fe(++) ion chelating ability. The total phenolic amount was also calculated as quite high (378.37 mg/g of gallic acid equivalents) in the crude ethanol extract. DISCUSSION AND CONCLUSION: Therefore, the obtained results tend to suggest the antinociceptive and antioxidant activities of the ethanol extract of the plant leaves and justify its use in folkloric remedies.

Effects of salt, BHA/BHT, and differing phosphate types on quality and sensory characteristics of beef longissimus muscles.

USDA Select striploins (n = 20) were cut into thirds (anterior, medial, and posterior) and randomly assigned to 1 of 6 treatments. Treatments included: (1) control (C); (2) 0.006% BHA (butylated hydroxyl anisole)/BHT (butylated hydroxytoluene) (70%/30%) (BB); (3) 0.4% trisodiumphosphate (CT); (4) 0.4% sodiumtripolyphosphate with 0.5% salt (BH); (5) sodiumtripolyphosphate, 0.5% salt, and 0.006% BHA/BHT (70%/30%) (SB); (6) 0.2% sodiumtripolyphosphate, 0.2% trisodiumphosphate, and 0.5% salt (STB). Muscle sections were injected to 110% (10% pump) of their weight with their respective treatments. Inclusion of BHA/BHT allowed for lower mean oxidation values. Regardless of phosphate type, muscles treated with both phosphate and salt had lower retail purge (P < 0.05). Sensory panelists rated (P < 0.05) STB, SB, and BH to be juicier than all other treatments. These data suggest that inclusion of both salt and phosphate can enhance palatability, lower cook loss, and retail purge.

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.

In vivo pharmacokinetics, activation of MAPK signaling and induction of phase II/III drug metabolizing enzymes/transporters by cancer chemopreventive compound BHA in the mice.

Phenolic antioxidant butylated hydroxyanisole (BHA) is a commonly used food preservative with broad biological activities, including protection against chemical-induced carcinogenesis, acute toxicity of chemicals, modulation of macromolecule synthesis and immune response, induction of phase II detoxifying enzymes, as well as its undesirable potential tumor-promoting activities. Understanding the molecular basis underlying these diverse biological actions of BHA is thus of great importance. Here we studied the pharmacokinetics, activation of signaling kinases and induction of phase II/III drug metabolizing enzymes/transporter gene expression by BHA in the mice. The peak plasma concentration of BHA achieved in our current study after oral administration of 200 mg/kg BHA was around 10 microM. This in vivo concentration might offer some insights for the many in vitro cell culture studies on signal transduction and induction of phase II genes using similar concentrations. The oral bioavailability (F) of BHA was about 43% in the mice. In the mouse liver, BHA induced the expression of phase II genes including NQO-1, HO-1, gamma-GCS, GST-pi and UGT 1A6, as well as some of the phase III transporter genes, such as MRP1 and Slcolb2. In addition, BHA activated distinct mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), as well as p38, suggesting that the MAPK pathways may play an important role in early signaling events leading to the regulation of gene expression including phase II drug metabolizing and some phase III drug transporter genes. This is the first study to demonstrate the in vivo pharmacokinetics of BHA, the in vivo activation of MAPK signaling proteins, as well as the in vivo induction of Phase II/III drug metabolizing enzymes/transporters in the mouse livers.

An ortho dimer of butylated hydroxyanisole inhibits nuclear factor kappa B activation and gene expression of inflammatory cytokines in macrophages stimulated by Porphyromonas gingivalis fimbriae.

Butylated hydroxyanisole, BHA, is widely used as a potent antioxidant, but its adverse effects such as carcinogenesis and proinflammatory activity have been reported, which are possibly due to the prooxidant property of this compound. We recently demonstrated that the dimer of 2-methoxyphenols exhibits cyclooxygenase-2 inhibition, because of lessening of its prooxidant property caused by the dimerization. In the present study, toward our goal of developing a chemopreventive agent for chronic periodontal diseases, we examined whether 2-BHA (2-tert-butyl-4-methoxyphenol) and its synthetic ortho dimer, bis-BHA (3,3'-di-tert-butyl-5,5'-dimethoxy-1,1'-biphenyl-2,2'-diol) could inhibit the Porphyromonas gingivalis fimbria-stimulated inflammatory reaction. The fimbria-induced expression of interleukin-1beta and neutrophil chemoattractant KC genes in RAW264.7 murine macrophages was strongly inhibited by bis-BHA. In contrast, 2-BHA showed only slight inhibition. bis-BHA also significantly inhibited the fimbria-stimulated phosphorylation-dependent degradation of the alpha inhibitor of nuclear factor-kappaB and the transcriptional activity of this factor in the cells. These findings suggest that bis-BHA possesses anti-inflammatory activity against chronic periodontal diseases.

Estimated daily intakes of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) antioxidants in Korea.

The study was conducted to establish the estimated daily intake (EDI) of antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) in Korea. The EDIs were obtained from two sources. One of the estimations was based on the analytical determination of BHA, BHT and TBHQ in 12 food categories (ten food categories for TBHQ) and on individual dietary intake data obtained from the National Health and Nutrition Survey in 1998 (n=11 525, age > 1 year). The other EDIs of BHA, BHT and TBHQ were based on the maximum permitted levels specified in national food standards in Korea and on individual dietary intake data obtained from the National Health and Nutrition Survey in 1998 (n=11 525, age > 1 year). To establish the EDIs based on the analytical determination and on individual dietary intake data, 133 food samples in 12 food categories were selected from the foods considered to be representative sources of BHA, BHT and TBHQ in the Korean diet. Selected samples were analysed by GC with FID. BHA was not detected in any of the samples analysed. BHT and TBHQ were detected in the samples, but the levels were significantly lower than their maximum limits. The EDIs1 of BHT, and TBHQ for average consumers were 0.0156(-3), and 0.0012(-3) mg kg(-1) body weight bw day(-1) and as a proportion of the ADI were 0.0052 and 0.0002%, respectively. For 95th percentile consumers, the EDIs of BHT and TBHQ were 0.0080 and 0.0006 mg kg(-1) bw day(-1), and as a proportion of the ADI were 2.67 and 0.09%, respectively. EDIs for BHA, BHT and TBHQ based on the maximum permitted levels and on individual dietary intake data were 0.04, 0.04 and 0.04 mg kg(-1) bw day(-1), respectively. The EDIs of BHA, BHT and TBHQ for average consumers ranged from 6.00 to 14.42% of the ADI of each antioxidant. According to these results, the EDIs of BHA, BHT and TBHQ in Korea were significantly lower than ADI of these antioxidants established by the JECFA.

Comparison of the amelioration effects of two enzyme inducers on the inflammatory process of experimental allergic encephalitis (EAE) using immunohistochemical technique.

Experimental Allergic Encephalitis (EAE) is a T-cell mediated autoimmune disease, which resembles the human disease Multiple Sclerosis (MS) in rodents. The infiltration of inflammatory cells and the induction of astrocyte proliferation correlate with EAE severity. Oxidative stress is postulated to have a role in the onset and progression of MS. Therefore, by reducing oxidative stress via phase II enzymes inducers; namely the butylhydroxyanisole (BHA) and Thymoquinone (glutathione inducer), the inflammation could be ameliorated. EAE was induced in Lewis rats using Myelin Basic Protein (MBP) and complete Freund's adjuvant (CFA). Animals were placed into 1. those on normal rat chow, 2. those on rat chow containing BHA, 3. those receiving concomitant five day injection of thymoquinone days 1-5 post-EAE induction, 4. those receiving five doses of thymoquinone injected at day 12-17 post-EAE induction. Twenty-nine days later, animals from each group were sacrificed and tissues collected for immunohistochemistry using the anti-glial fibrillary acid (GFAP) antibody to examine the amelioration effect these two agents have on the inflammatory process occurring in EAE by examining the astrocyte proliferation in the Central Nervous System (CNS).

Increased microbicidal activity of green tea (Camellia sinensis) in combination with butylated hydroxyanisole.

We have demonstrated that green tea (Camellia sinensis) shows increased antimicrobial activity against bacteria and fungi when used in combination with butylated hydroxyanisole (BHA). Glycolic extract taken from green tea showed only limited activity against Streptococcus mutans and no activity against Candida albicans and certain strains of Escherichia coli. BHA, at non inhibitory concentrations, increased the microbicidal activity of green tea against 10(10) S. mutans (p<0.01), non-susceptible E. coli (p<0.01) and C. albicans (p<0.01). Green tea in combination with BHA reduced the hydrophobicity of S. mutans (p<0.01) and greatly inhibited (p<0.001) the formation of hyphae in C. albicans. The increased antimicrobial activity of green tea is related to an impairment of the barrier function in microorganisms and a depletion of thiol groups. The increased activity of green tea as an oral antimicrobial product is discussed.

Molecular mechanism of cell death induced by the antioxidant tert-butylhydroxyanisole in human monocytic leukemia U937 cells.

A phenolic antioxidant 3-tert-butyl-4-hydroxyanisole (BHA) is a widely used food additive. BHA had cytotoxicity in human monocytic leukemia U937 cells. BHA at 0.75 mM caused nuclear condensation and fragmentation, structural damage in mitochondria, decrease in mitochondrial transmembrane potential, and internucleosomal DNA cleavage. It induced the activities of caspase-3 and/or -7, -6, -8 and -9, especially high when DEVD-MCA was the substrate (caspase-3 and/or -7). DEVDase activity increased in time- and dose-dependent manner and high activity was observed in lysates of cells treated for 3 h at 0.75 mM. Addition of GSH (reduced glutathione) during the treatment of cells with BHA inhibited the induction of DEVDase activity, and the intracellular GSH level decreased as the concentration of BHA was raised. Intracellular ATP levels decreased in time- and dose-dependent manner when the cells were treated with BHA in the presence or absence of glucose. Enzyme activities involved in the respiratory chain were assayed with the mitochondrial fraction prepared from U937 cells. BHA distinctly inhibited NADH-ubiquinone oxidoreductase (complex I) and cytochrome c oxidase (complex IV) at low concentrations. Succinate-ubiquinone oxidoreductase (complex II) was also inhibited, but to somewhat less extent. Without mitochondrial enzymes, BHA stimulated the ubiquinol-dependent reduction of cytochrome c (complex III), but it might have some detrimental effects on the mitochondrial enzyme reaction of complex III. The inhibition of mitochondrial oxidative phosphorylation might corroborate the mechanistic evidence for apoptosis of leukemia cells by BHA. Cell death induced by BHA is primarily ascribable to apoptosis.

Effect of sumach (Rhus coriaria L.) extracts on the oxidative stability of peanut oil.

The antioxidant activities of sumach (Rhus coriaria L.) extracts and butylated hydroxyanisole (BHA) at various concentrations were tested in natural peanut oil stored at 65 degrees C for 35 days. The concentrations (weight/volume) of extracts added into oil were 1.0%, 3.0%, and 5.0%, and those of BHA were 0.1%, 0.3%, and 0.5%. Antioxidant effect was determined by the measurement of peroxide value. After 7 days of storage, BHA and extracts of sumach were active in varying degrees against autoxidation of peanut oil, compared with the control test (P <.01). The sumach extracts generally inhibited the formation of hydroperoxide, as did BHA. After 28 days of storage, antioxidant effects of extracts were significantly decreased when compared with BHA. The decrease in the antioxidant activity of extracts might have resulted from the decrease of polyphenolic constituents. The results showed that high concentrations can enhance the potency of the antioxidant effect of sumach extract.

Amelioration of experimental allergic encephalitis (EAE) through phase 2 enzyme induction.

The multiple sclerosis (MS) lesion is characterized by an inflammatory cell mediated attack on white matter. Oxidative stress appears to play a role in the onset and progression of MS. We reasoned that decreasing oxidative stress might ameliorate MS. One way of decreasing oxidative stress is to induce phase 2 enzymes. The model chosen to test this hypothesis was experimental allergenic encephalomyelitis (EAE) induced in the Lewis rat. The 26 animals were placed into two groups: 1) those on normal rat chow, 2) those on rat chow containing 250 mumoles t-butylhydroxyanisole (BHA)/kg. After 2 weeks, animals were administered 100 micrograms guinea pig myelin basic protein and examined daily in a blinded fashion. Twenty-nine days later, animals were sacrificed, blood collected for glutathione (GSH) measurements and tissues collected for histology. Six of the 13 control chow animals developed hindlimb weakness or paralysis while 5 developed tail weakness only. Only 1 BHA fed animal exhibited symptoms--hindlimb weakness. Clinical symptoms correlated well with the extent of perivascular lymphocyte infiltration. Animals with BHA in the diet had 20% higher red cell GSH indicting induction of phase 2 enzymes. We conclude that dietary phase 2 enzyme inducers should be examined for their ability to ameliorate MS.

Effects of butylated hydroxyanisole and butylated hydroxytoluene on DNA adduct formation and arylamine N-acetyltransferase activity in human bladder tumour cells.

In this study, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were used to determine the inhibition of arylamine N-acetyltransferase (NAT) activity and DNA adduct formation in human bladder tumour cell line T-24. The activity of NAT was measured by high-performance liquid chromatography, assaying for the amounts of N-acetyl-2-aminofluorene and N-acetyl-p-aminobenzoic acid and remaining 2-aminofluorene and p-aminobenzoic acid. Human bladder tumour cell line T-24 cytosols and intact cells were used for examining NAT activity and carcinogen-DNA adduct formation. The results demonstrated that NAT activity and 2-aminofluorene-DNA adduct formation in human bladder tumour cells were inhibited and decreased by BHA and BHT in a dose-dependent manner. The effects of BHA and BHT on the values of the apparent K(m) and V(max) also were determined in both systems examined. The results indicated that BHA and BHT decreased the apparent values of K(m) and V(max) from human bladder tumour cells in both cytosol and intact cells.

Estimates of the theoretical maximum daily intake of phenolic antioxidants BHA, BHT and TBHQ in Brazil.

The theoretical maximum daily intakes (TMDI) of the phenolic antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertbutyl hydroquinone (TBHQ) in Brazil were estimated using food consumption data derived from a household economic survey and a packaged goods market survey. The estimates were based on maximum levels of use of the food additives specified in national food standards. The calculated intakes of the three additives for the mean consumer were below the ADIs. Estimates of TMDI for BHA, BHT and TBHQ ranged from 0.09 to 0.15, 0.05 to 0.10 and 0.07 to 0.12 mg/kg of body weight, respectively. To check if the additives are actually used at their maximum authorized levels, analytical determinations of these compounds in selected food categories were carried out using HPLC with UV detection. BHT and TBHQ concentrations in foodstuffs considered to be representive sources of these antioxidants in the diet were below the respective maximum permitted levels. BHA was not detected in any of the analysed samples. Based on the maximal approach and on the analytical data, it is unlikely that the current ADI of BHA (0.5 mg/kg body weight), BHT (0.3 mg/kg body weight) and TBHQ (0.7 mg/kg body weight) will be exceeded in practice by the average Brazilian consumer.

Induction of hepatic DT-diaphorase in mice by butylated hydroxyanisole analogs: a structure-activity study.

Butylated hydroxyanisole (BHA) and its analogs were evaluated for their relative activity to induce hepatic DT-diaphorase (EC 1.6.99.2) after dietary administration (at concentrations of 11.1 or 27.7 micromol/g diet for 3 days) to mice. Of the compounds tested, only BHA and 2-tert-amyl-4-methoxyphenol, 4-methoxyphenols with 2-tert-alkyl side chains, were active in inducing DT-diaphorase activity. None of the remaining six compounds showed any significant sign of inducing activity. No simple explanation for these rather strict structural requirements can be offered at the present time.

Estimates of the theoretical maximum daily intake of erythorbic acid, gallates, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) in Italy: a stepwise approach.

The three recent EU directives which fixed maximum permitted levels (MPL) for food additives for all member states also include the general obligation to establish national systems for monitoring the intake of these substances in order to evaluate their use safety. In this work, we considered additives with primary antioxidant technological function for which an acceptable daily intake (ADI) was established by the Scientific Committee for Food (SCF): gallates, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and erythorbic acid. The potential intake of these additives in Italy was estimated by means of a hierarchical approach using, step by step, more refined methods. The likelihood of the current ADI to be exceeded was very low for erythorbic acid, BHA and gallates. On the other hand, the theoretical maximum daily intake (TMDI) of BHT was above the current ADI. The three food categories found to be main potential sources of BHT were "pastry, cake and biscuits", "chewing gums" and "vegetables oils and margarine"; they overall contributed 74% of the TMDI. Actual use of BHT in these food categories is discussed, together with other aspects such as losses of this substance in the technological process and percentage of ingestion in the case of chewing gums.

Induction of murine intestinal and hepatic peroxiredoxin MSP23 by dietary butylated hydroxyanisole.

Feeding mice with 2(3)-t-butyl-4-hydroxyanisole (BHA) induces phase II detoxifying enzymes that inhibit the action of carcinogens. We have found that dietary BHA induces intestinal and hepatic MSP23 (also called peroxiredoxin I), a stress-inducible antioxidant, in a manner similar to the induction of glutathione S-transferases (GSTs). The levels of MSP23 in the proximal intestine and liver, estimated by immunoblotting, increased approximately 1.9- and 1.3-fold, respectively, in mice fed a diet containing 0.7% (w/w) BHA for 7 days. The level of MSP23 mRNA in these tissues also increased more than 2-fold after mice were fed BHA, suggesting that the induction of MSP23 is controlled at the transcription level. Immunostaining of the small intestine shows that MSP23 is expressed mainly in the columnar epithelial cells. The induction of MSP23 may be important to protect the cells and tissues against toxic electrophiles and reactive oxygen species.

Antioxidants stimulate meiosis resumption, but inhibit mitogen-activated protein kinase phosphorylation and further cell cycle progression in porcine oocytes.

In the present study the effects of two cell-permeant antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and nordihydroguaiaretic acid (NDGA), on porcine oocyte meiosis resumption, chromatin behaviour and spindle assembly were investigated. The antioxidants BHA and NDGA stimulated meiosis resumption in a dose-dependent manner in both cumulus-enclosed and denuded porcine oocytes. After in vitro culture for 8 h, few oocytes underwent germinal vesicle breakdown (GVBD) in control groups, whereas GVBD occurred in high percentages of oocytes treated with BHA or NDGA at concentrations that inhibit GVBD in rodent oocytes, although mitogen-activated protein (MAP) kinase was not phosphorylated as revealed by Western immunoblots. Orcein staining and fluorescein isothiocyanate-anti-alpha-tubulin labelling showed that chromosome and spindle formation, respectively, and further meiosis progression were inhibited 20 and 25 h after culture. Instead, chromatin was highly condensed or existed in scattered condensed clusters. Correspondingly, MAP kinase phosphorylation was inhibited by both BHA and NDGA in a dose-dependent manner. The inhibitory effects of BHA on meiosis completion and MAP kinase phosphorylation was reversible. These results suggest that, unlike in rodent oocytes, antioxidants stimulate GVBD in the absence of MAP kinase activation, but inhibit MAP kinase phosphorylation, meiotic apparatus formation and thus the further progression of the meiosis of porcine oocytes.