Dietary butylated hydroxytoluene improves lipid metabolism, antioxidant and anti-apoptotic response of largemouth bass (Micropterus salmoides).

A 10-week growth trail was conducted to investigate the efficacy and tolerance of dietary butylated hydroxytoluene (BHT) by evaluating inflammation, apoptosis and hepatic disease related to oxidative stress in largemouth bass (Micropterus salmoides). Four experimental diets were prepared with BHT supplement levels of 0 (B0), 150 (B150), 300 (B300) and 1500 (B1500) mg/kg, in which B150 was at the maximum recommended level established by European Union Regulation, and the B300 and B1500 levels were 2 and 10-fold of B150, respectively. Each diet was fed to 6 replicates with 30 largemouth bass (initial body weight, IBW = 6.20 ± 0.01 g) in each tank. The BHT inclusion level did not affect the specific growth rate, but fish in the B150 group showed the lowest feed conversion rate (P < 0.05). BHT inclusion significantly decreased the levels of plasma TC, TG, LDL, ALT and AKP, and increased the (HDL-C)/TC ratio (P < 0.05). Plasma MDA was significantly decreased in the B150 group and GSH-Px was extremely enhanced in each BHT inclusion group (P < 0.05). Hepatic T-AOC was significantly enhanced and O2- was significantly decreased in each BHT inclusion group compared to the B0 group (P < 0.05), as well as hepatic MDA was significantly decreased in B1500 group (P < 0.05). Dietary BHT inclusion down-regulated the hepatic mRNA levels of inflammation, apoptosis and fibrosis related genes, including TNFα, TGF-ß1, α-SMA, IL8, IL11ß and caspase-9. Moreover, BHT could improve hepatic lipid metabolism via up-regulating the mRNA levels of APOA1, CYP7A1, CYP8B1, and down-regulating the mRNA levels of PPAR-γ and APOB. Histological examination of the liver morphology with H&E and Sirius Red staining showed that BHT inclusion decreased necrotic degenerative changes and collagen deposition in largemouth bass. An immunofluorescence examination revealed significantly decreased cleaved caspase-3 signals in the BHT groups. In conclusion, the results demonstrated that ROS induces hepatic cell apoptosis and fibrosis via the intrinsic pathway of apoptosis by activating caspase-9 in the mitochondria and then initiates apoptosis by activating caspase-3. Consuming 2.32-23.80 mg/kg·bw/d (150-1500 mg/kg in diet) of BHT effectively improved the plasma and hepatic lipid metabolism, antioxidant response as well as reduced ROS production, protecting hepatic cells from injury. It is implied that even a 10-fold increase of the maximum level of BHT (150 mg/kg) is safe for the largemouth bass.

Butylated hydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo (Bubalus bubalis).

The study was carried out to evaluate the potential impact of butylated hydroxytoluene (BHT) on the frozen-thawed semen quality of Nili-Ravi buffalo bulls. Ejaculated bull semen was extended in a Tris-citrate egg yolk extender containing various concentrations of BHT (0.5, 1.0, 2.0 and 3.0mM). Semen was frozen at -196 degrees C using 50 x 10(6) spermatozoa per 0.5 mL straws. Five straws from each treatment were thawed to assess the semen quality in terms of sperm motility, viability, plasma membrane integrity and acrosomal integrity. Post-thawed sperm motility was determined using a phase-contrast microscope. Viability, plasma membrane integrity and acrosomal integrity were evaluated by the supravital staining, hypo-osmotic swelling test and normal acrosomal reaction, respectively. The highest (P<0.05) motility, acrosomal integrity and hypo-osmotic swelling response of spermatozoa was achieved by addition of 1.0 and 2.0mM BHT to semen extender. However, highest (P<0.05) viability of spermatozoa was achieved by inclusion of 2.0mM BHT. The higher concentration of BHT (3.0mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of the spermatozoa compared to other concentration used. In conclusion, BHT when added in the semen extender can improve the semen quality of buffalo bulls.

Effect of the flavonoid baicalein as a feed additive on the growth performance, immunity, and antioxidant capacity of broiler chickens.

Baicalein, the main flavonoid extracted from the root of Scutellaria baicalensis Georgi, has been demonstrated to exert multiple pharmacological effects, and thus could be utilized as a potential feed additive in broiler chickens. This study evaluated the effects of broiler chicken diet supplementation with baicalein on growth performance, immunity, and antioxidant activity at levels of 100 and 200 mg/kg. No significant effect on average daily feed intake (P > 0.05) of broilers with diets supplemented with baicalein was found compared to those on the basal diet or butylated hydroxytoluene (BHT) during the 35-d feeding trial. The addition of baicalein to the basal diet significantly increased average body weight, body weight gain, average weight gain, and the feed conversion ratio of birds during 21 to 42 d and 7 to 42 d of age, respectively. The best numerical values for the overall growth performance were observed in broilers fed on diets containing 200 mg/kg of baicalein. Baicalein supplementation significantly increased the ratio of CD3+/CD4+ and CD3±/CD8+, the concentration of IFN-γ, anti-IB antibody titer, and the spleen index compared with the control group (P < 0.05). Total cholesterol, the ratio of non-HDL-C/HDL-C, LDL-C/HDL-C, TC/HDL-C, triglycerides, and low-density lipoprotein cholesterol were significantly decreased after intake of baicalein compared with both the basal diet and the BHT-supplemented diet, whereas the SOD, GSH-Px, and CAT activity in the serum increased with the supplementation of baicalein. The T-AOC activity, T-SOD, and GSH-Px level in liver tissues was significantly increased by inclusion of baicalein, and intake of baicalein or BHT significantly decreased the malondialdehyde level found both in serum and meat tissue. Thus, the results obtained here indicate that the baicalein can be used as an effective natural feed additive in broiler chicken diets, and that 100 to 200 mg/kg can be considered as the optimum dosage.

Effect of butylated hydroxyl toluene on the immune response of Rift Valley fever vaccine in a murine model.

The present study was planned to examine the effect of butylated hydroxy toluene (BHT) on the immune response of Rift Valley fever vaccine (RVFV) in Swiss mice. Animals were divided into four equal groups. The first group was kept as negative control. The 2nd group was orally administrated with the acceptable daily intake (ADI) of BHT 0.3 mg/kg b.wt. daily for 21 days and the 3rd group were vaccinated only by inactivated RVFV at a dose of 0.2 ml I/P two times. The 4th group was orally administrated BHT as in the 2nd group and vaccinated by inactivated RVFV as in the 4th group. Blood samples were collected from all groups two weeks from booster vaccination. The cellular immunity was determined by leucocytic indices and the neutrophil-lymphocyte ratio (NLR) whereas, humoral immunity was evaluated with IgG antibodies titer using enzyme-linked immune-sorbent assay (ELISA) test, serum neutralization test (SNT) and challenge test. BHT induced leucopenia, neutrophilia and marked lymphocytopenia in both non-vaccinated and vaccinated mice. Moreover, BHT significantly decreased the efficiency of vaccination by inducing 70% cytopathic effect (CPE) in the infected cell cultures and increasing the ED50 value of RVFV vaccine. The present study indicates that BHT possesses a potential for decreasing both cellular and humoral mediated mechanisms.

Role of maternal dietary antioxidant supplementation in murine placental and fetal limb development.

Methylnitrosourea (MNU) is a multisystem teratogen that damages proliferating cells through macromolecule alkylation and generation of reactive oxygen species (ROS). Murine dams exposed to MNU midgestation produce offspring with distal limb malformations, an outcome reduced by maternal immune stimulation. Immunostimulatory effects of antioxidant therapy may in part explain this improved birth outcome. The present study hypothesizes that placental, rather than fetal, damage from excessive ROS may contribute to MNU-induced embryopathy. Fetal limbs and placentas were examined in immunotolerant CD-1 and immunosensitive C57BL/6N mice exposed to MNU, dietary antioxidant butylated hydroxytoluene (BHT), or both. MNU increased fetal resorptions and incidence of syndactyly, oligodactyly, polydactyly, and interdigital webbing, and decreased fetal size in both mouse strains. BHT reduced syndactyly and oligodactyly in both strains, and reduced polydactyly in C57BL/6N mice. Increased webbing in MNU and MNU+BHT groups likely represented maturational delay. Placentas from CD-1 and C57BL/6N MNU-exposed dams demonstrated decreased trophoblasts and increased necrosis of endothelium. Similar to distal limb defects, placental damage was reduced in mice receiving MNU+BHT. These results suggest that placental damage and fetal defects caused by MNU are in part ROS-mediated, and reduced distal limb defects following MNU+BHT may be related to improved placental integrity and function.

Inhibition of chemically induced mammary carcinogenesis in rats by short-term exposure to butylated hydroxytoluene (BHT): interrelationships among BHT concentration, carcinogen dose, and diet.

Dietary butylated hydroxytoluene (BHT) fed 14 days before and 14 days after carcinogen administration resulted in a dose-dependent inhibition of 7, 12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor incidence in outbred Sprague-Dawley rats. In addition, the inhibitory effects of BHT were strongly influenced by the dose of initiating carcinogen and the type of diet in which BHT was administered. In animals fed the NIH-07 diet and receiving a low dose of DMBA (5 mg/rat), the inhibitory effect of BHT was manifested at all four BHT concentrations (6,000 leads to 300 ppm). Maximal inhibition was approximately 50% in animals given 5 mg DMBA and receiving 6,000 ppm BHT. However, in the group administered a high dose of DMBA (15 mg/rat), the inhibitory effect of BHT was expressed only at 6,000 ppm, the highest concentration given. Lower concentrations (300 and 1,000 ppm) of BHT had no detectable effect on tumor incidence. In animals fed the defined, semipurified AIN-76A diet during the 4-week treatment period and initiated with 5 mg DMBA, BHT at 6,000 ppm inhibited tumor development. However, at 15 mg DMBA animals fed the AIN-76A diet differed markedly from those fed the NIH-07 diet. In the former group, BHT at 6,000 ppm was unable to elicit any inhibitory response; in the latter group, BHT inhibited tumor development by 40%. Dietary BHT also inhibited DMBA-induced adrenocortical hyperplastic nodules in a dose-dependent fashion. These results indicate that short-term exposure to dietary BHT can inhibit experimental mammary tumor development at environmentally relevant concentrations.

Inhibition of chemically induced mammary carcinogenesis in rats by long-term exposure to butylated hydroxytoluene (BHT): interrelations among BHT concentration, carcinogen dose, and diet.

In outbred female Sprague-Dawley rats long-term exposure to dietary butylated hydroxytoluene [3,5-di-tert-butyl-4-hydroxytoluene (BHT); CAS: 128-37-0] 1 week before carcinogen administration to termination resulted in a dose-related inhibition of mammary tumorigenesis and adrenocortical nodulogenesis. In animals fed the cereal-based NIH-07 diet and receiving a low dose (5 mg/rat) of 7,12-dimethylbenz [a] anthracene [(DBMA) CAS: 57-97-6], there was a significant overall inhibitory trend in tumor incidence observed among those receiving 300, 1,000, 3,000, and 6,000 ppm BHT. Maximal inhibition was approximately 50% at the highest concentration of BHT (6,000 ppm). The inhibitory effect of BHT on mammary tumor incidence was less pronounced when BHT was administered to rats initiated with a high carcinogen dose: At 15 mg DMBA/rat maximal inhibition was only 20% at the highest concentration of BHT (6,000 ppm). In contrast, when tumor yield was assessed in terms of latency or tumor multiplicity, the inhibitory effect of BHT was more pronounced in the groups given a high dose of DMBA than in the groups given a low dose. In animals given a low dose of DMBA (5 mg) and fed 6,000 ppm BHT in the casein-based AIN-76A diet, tumor incidence was inhibited by 50% of that of the controls; in contrast, when initiation was with a high dose of DMBA (15 mg), tumor incidence was decreased by only 28% of that of the controls. In animals fed the NIH-07 diet, DMBA-induced adrenocortical nodule formation was also inhibited in a dose-dependent fashion by BHT. At 5 mg DMBA maximal inhibition was 86% of control levels (6,000 ppm BHT); at 15 mg DMBA maximal inhibition was 66% of control levels (6,000 ppm BHT). However, when BHT was incorporated into the AIN-76A diet, its inhibitory effects on adrenocortical nodulogenesis were unexpectedly feeble and unrelated to carcinogen dose: In animals initiated with 5 mg DMBA and administered 6,000 ppm BHT, nodule incidence was decreased by only 25%, whereas in animals initiated with 15 mg DMBA, nodule incidence was decreased by 30% of that of the controls. These results indicate that while chronic exposure to dietary BHT suppressed the development of DMBA-induced mammary tumors and adrenocortical nodules, the degree of suppression depended on the dose of carcinogen administered, the level of BHT in the diet, and the parameter being measured. Diet-dependent differences in BHT action were observed with regard to DMBA-induced adrenocortical nodulogenesis but not with regard to mammary tumorigenesis.

Enhancement of urethan tumorigenesis in mouse lung by butylated hydroxytoluene.

Intraperitoneal injection of the antioxidant butylated hydroxytoluene (BHT) produces cell proliferation in mouse lungs within 2-4 days. We examined whether the presence of an increased number of proliferating lung cells would influence urethan tumorigenesis. Male Swiss-Webster mice were treated with 1 mg urethan/g before, during, or after BHT-stimulated cell growth in the lung. The number of pulmonary tumors found 13-15 weeks later was not different in BHT-treated mice compared to that in controls. On the other hand, repeated stimulation of cell growth after urethan treatment enhanced tumorigenesis. Male Swiss-Webster and A/J mice were given a single dose of urethan (1 mg/g) and, beginning 7 days later, weekly injections of BHT or corn oil. Repeated injections of BHT significantly increased the yield of lung tumors in both strains. Weekly injections of BHT into mice pretreated with 0.9% NaCl reduced the number of spontaneous pulmonary adenomas.

Combination chemoprevention of rat mammary carcinogenesis by indomethacin and butylated hydroxytoluene.

Indomethacin, a nonsteroidal antiinflammatory agent which inhibits prostaglandin biosynthesis, is an effective inhibitor of mammary carcinogenesis in rats. However, the activity of indomethacin as a chemopreventive agent is limited by toxicity. The present studies were conducted to determine if the toxic and anticarcinogenic effects of indomethacin can be modified by the phenolic antioxidant, butylated hydroxytoluene (BHT). Simultaneous administration of BHT resulted in a dose-related inhibition of indomethacin toxicity in female Sprague-Dawley rats, and increased the tolerable indomethacin dose from 50 to 150 mg/kg diet. When BHT (5000 mg/kg diet) and indomethacin (50 mg/kg diet) were administered in combination, no increased inhibition of 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis was observed above that attained by administration of BHT alone or indomethacin alone at those doses. However, when the indomethacin dose was increased to 100 mg/kg diet, an enhanced inhibition of carcinogenesis was attained when BHT and indomethacin were administered from 2 weeks prior to until 1 week after 7,12-dimethylbenz(a)anthracene administration. These data indicate that "combination chemoprevention" regimens can be utilized to reduce the toxicity of anticarcinogenic drugs. However, the BHT-indomethacin interaction appears to involve a functional or dispositional antagonism which limits the anticarcinogenic efficacy of increasing indomethacin dose level.

Inhibition of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinogenesis by concomitant or postcarcinogen antioxidant exposure.

When administered prior to or at the time of carcinogen exposure, the phenolic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are effective inhibitors of carcinogenesis in several target organs. However, chronic, postcarcinogen administration of BHT apparently enhances tumorigenesis in certain animal models for liver and lung cancer. The present study was performed to determine the effects of BHA and BHT on mammary carcinogenesis when antioxidant exposure is limited to defined periods encompassing or following carcinogen availability. At 50 days of age (Time O), virgin female Sprague-Dawley rats (25/group) were given a single intragastric dose of 8 mg of 7,12-dimethylbenz(a) athracene . Basal diet (Wayne Lab Meal) was supplemented with 5000 or 2500 mg of BHA or BHT/kg by the following protocol: 2 weeks before until 1 week after carcinogen administration; 1 week after carcinogen administration until the end of the study; or none. The experiment was terminated 210 days after 7,12-dimethylbenz(a)anthracene administration, and all mammary tumors were confirmed histologically. When administered by the 2 weeks before to 1 week after schedule, both BHA and BHT were effective inhibitors of mammary carcinogenesis. However, the compounds also were active in chemoprevention when administered by the 1 week after to end protocol. These data indicate that the anticarcinogenic activity of antioxidants is not limited to influences on carcinogen metabolism, since both BHA and BHT inhibited mammary tumor induction when their administration was begun following clearance of the carcinogen from the mammary gland. The anticarcinogenic activity of postcarcinogen administration of BHA and BHT in the mammary gland is in contrast to the apparent tumor-enhancing activity of BHT in the liver and lung.

Anticarcinogenic and hepatotoxic interactions between retinyl acetate and butylated hydroxytoluene in rats.

The natural retinoid, retinyl acetate (RA), and the phenolic antioxidant, butylated hydroxytoluene (BHT), are both effective inhibitors of mammary carcinogenesis in rats. The present study was designed to determine if an increased inhibition of mammary carcinogenesis is obtained when RA and BHT are administered in combination. At age 50 days (time 0), virgin, female Sprague-Dawley rats received a single intragastric instillation of 16 mg of 7,12-dimethylbenz(a)anthracene dissolved in 1 ml sesame oil. Groups of 30 carcinogen-treated rats received Wayne Lab Chow supplemented with (per kg diet) 250 mg RA, 5000 mg BHT, or 250 mg RA plus 5000 mg BHT by the following schedule: -2 to +1 week; +1 week until the end of the experiment; -2 weeks to end; or none. Combined administration of RA plus BHT by the -2 weeks to end schedule was more effective in mammary cancer chemoprevention than was RA alone or BHT alone; the interaction of RA and BHT was additive. Similarly, administration of RA plus BHT by the -2 weeks to end protocol was more active in chemoprevention than was RA plus BHT administered either from weeks -2 to +1 or +1 week to end. Chronic exposure to RA plus BHT induced a high incidence of hepatic fibrosis and bile duct hyperplasia; these changes were not observed in controls and were seen in low incidence in animals exposed to RA only or BHT only. These data indicate that enhanced anticarcinogenic activity can be obtained through the use of "combination chemoprevention" regimens; however, chemopreventive compounds may interact not only to inhibit carcinogenesis but also to induce toxicity.

Modulation by butylated hydroxytoluene of liver and bladder carcinogenesis induced by chronic low-level exposure to 2-acetylaminofluorene.

The modulating effect of five dose levels of butylated hydroxytoluene (BHT) on liver and bladder carcinogenesis induced in rats by concurrent exposure to 2-acetylaminofluorene (AAF) was investigated. AAF at a low dose of 50 ppm was fed simultaneously with concentrations of 100, 300, 1000, 3000, or 6000 ppm BHT in the diet to male F344 rats for up to 76 weeks. By 12 weeks, AAF alone induced altered hepatocellular foci, identified by iron storage deficiency and gamma-glutamyltranspeptidase activity. At subsequent time points of 24, 36, and 48 weeks, the number of foci progressively increased, and at the end of the study, the incidence of liver neoplasms was 100%, a new finding with such a low dose of AAF. Simultaneous feeding of BHT inhibited the induction of liver altered foci by AAF in a dose-related manner and reduced the incidence of hepatocellular carcinomas and the number of liver neoplasms per animal. Feeding of 6000 ppm BHT, but not of lower doses, together with AAF resulted in an increase in the incidence and multiplicity of bladder neoplasms, and 3000 ppm increased nodular hyperplasia of the bladder. These results suggest that the chemoprevention by BHT of cancer resulting from low-level long-term carcinogen exposure may be achieved at doses that do not produce adverse effects.

Comparative effects of dietary administration of 2(3)-tert-butyl-4-hydroxyanisole and 3,5-di-tert-butyl-4-hydroxytoluene on several hepatic enzyme activities in mice and rats.

Effects of feeding mice and rats with 2(3)-tert-butyl-4-hydroxyanisole (BHA) and 3,5-di-tert-butyl-4-hydroxytoluene (BHT), the two most commonly used food-additive phenolic antioxidants with known anticarcinogenic properties but with only minor differences in their chemical structures, have been compared to search for common effects between the two agents in two different rodent species and then applied toward better understanding of the mechanisms involved in their protective actions. In liver microsomes of treated mice, both BHA and BHT enhanced the relative activity of aniline ring hydroxylation but decreased the relative benzo(a)pyrene monooxidase activities. However, in rats, although aniline ring hydroxylation activity was decreased by both compounds, the decrease of benzo(a)pyrene monooxidase activity was observed only with BHT. Thus, common effects could not be recognized at the microsomal mixed-function oxidase level. Contrary to expectations based on chemical structures, BHT feeding elevated by epoxide hydrolase activity to an even greater extent than that produced by BHA, especially in rats. However, enzyme activities involved in the glucuronide conjugation system (uridine diphosphate:glucuronyl transferase, uridine diphosphate:glucose dehydrogenase, and quinone reductase) are all elevated by both antioxidants in both rodent species. With BHA treatment, the levels of acid-soluble thiols were increased in both rats and mice. However, with BHT, the level was increased only in mice but not in rats. Similar trends were produced for glucose-6-phosphate dehydrogenase activity, but glutathione reductase activity was increased even for BHT-treated rats. Additionally, the glutathione S-transferase activities were also increased by both antioxidant treatments and in both rodent species. Based on these results, the elevations of epoxide hydrolase activity along with the enhanced glucuronide conjugation and glutathione oxidation and reduction conjugation system enzyme activities were common to both compounds in both rodent species. This suggests their involvement in anticarcinogenic mechanisms. Increases of these detoxification enzyme activities appeared to be all designed to accelerate the elimination of administered antioxidants but, inadvertantly, conferring protective effects from xenobiotics such as carcinogens.

Activation of liver X receptor inhibits the development of pulmonary carcinomas induced by 3-methylcholanthrene and butylated hydroxytoluene in BALB/c mice.

We previously reported that LXR ligand, T0901317, inhibited the growth of inoculated Lewis lung carcinoma in C57BL/6 mice by activating IFN-γ production. However, the effects of T0901317 on carcinogen-induced pulmonary carcinomas remain unknown. In this study, we initially conducted a statistical analysis on the data of human lung cancer samples extracted from the TCGA database, and determined that survival rate/time of lung cancer patients and grade of lung adenocarcinoma were positively and negatively related to lung IFN-γ levels, respectively. We then determined the inhibitory effects of T0901317 on mouse pulmonary carcinomas induced by 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) or urethane. We found that T0901317 reduced morbidity and mortality in MCA/BHT-injected BALB/c mice by inhibiting lung adenocarcinoma. T0901317 also protected C57BL/6 mice, but not IFN-γ deficient (IFN-γ(-/-), C57BL/6 background) mice, against MCA/BHT-induced lung hyperplasia/inflammation. In addition, we determined that T0901317 inhibited urethane-induced lung tumors in BABL/c mice. Furthermore, we determined that T0901317 prevented metastasis of 4T1 breast cancer cells in BALB/c mice. Administration of T0901317 substantially increased serum IFN-γ levels and lung IFN-γ expression in BABL/c and C57BL/6 mice. Taken together, our study demonstrates that LXR inhibits MCA/BHT-induced pulmonary carcinomas in BABL/c mice and the inhibition is associated with induction of IFN-γ production.

[Changes in composition of acid soluble proteins and DNA in chromatin of rat liver and brain bound and not bound to nuclear envelope as a function of age and under the influence of antioxidant ionol].

In two-day rat pups, the histone H1 content in the brain chromatin was higher than in the liver chromatin, as compared to histone of the nucleosome core. The H1 content in the brain chromatin decreased with the age, while in the liver chromatin it increased. At the same time, in the adult brain chromatin bound to the nuclear envelope, a high level of H1 characteristic of chromatin of the newborn rats was preserved, while in a similar chromatin of the adult liver, the H1 content increased, but still remained less than in the chromatin not bound to the nuclear envelope. In both organs, the composition and quantitation of H1 subfractions were different in chromatins bound and not bound to the nuclear envelope. The chromatin from the liver and brain bound to the nuclear envelope differed also in the composition and quantitation of minor acid soluble proteins. In the presence of the antioxidant ionol, the 5-methylcytosine content in DNA of chromatin of the rat liver bound to the nuclear envelope increased while in the chromatin not bound to the nuclear envelope, it remained unchanged. Thus the chromatins bound and not bound to the nuclear envelope differ in the composition and mount of acid soluble proteins, including histone H1, the contents of these proteins in bound and not bound chromatin are different and change with the age in different ways. The antioxidant ionol affects differently the methylation of bound and not bound chromatin.

[Activation of deoxyribonucleotide synthesis by radioprotectants and antioxidants as a key stage in formation of body resistance to DNA-damaging factors].

The responses of the systems of synthesis of deoxyribonucleotides (dNTPs), DNA, and proteins in hematopoietic organs and liver of animals to gamma-radiation, administration of radioprotectants and antioxidants as well as the dependence of these responses on the doses of radiation and drugs were studied. Radioprotectants of acute (indralin) and durable effects (indomethaphen) as well as natural (alpha2-tocopherol) and synthetic anti-oxidants (ionol or 2,6-di-tert-butyl-4-methylphenol) efficient in survival test were used. Three stages could be recognized in the standard unspecific response of the studied systems to radiation: (1) immediate increase in ribonucleotide reductase activity in the tissues within the first 30 min as a part of the integrated SOS response to DNA damage, which activates dNTP synthesis; (2) inhibition of the synthesis of dNTPs, DNA, and and (3) restoring ribonucleotide reductase activity and integral increase in the production of dNTPs, DNA, and total protein, which is essential for the development of compensatory and restorative responses of the organism. The radioprotectants significantly increased ribonucleotide reductase activity, which increased intracellular concentrations of the four dNTP types in organs during radiation exposure and three following days. Within this period, ribonucleotide reductase activity was inhibited by 40-50% in animals not treated with radioprotectants as compared to control. Balanced high pools of dNTPs in the organs of radioprotectant-treated animals provided for high-performance repair of DNA damage. The radioprotectant-induced activation of dNTP synthesis during the development of compensatory and restorative responses provides for an earlier restoration of the cellular composition and functioning of the organs. Antioxidants stimulated the synthesis of dNTPs, DNA, and proteins in animal tissues in a strict dose interval. Their effect on the studied syntheses was dose-dependent: single or multiple long-term administration of high antioxidant doses inhibited synthesis of dNTPs, DNA, and proteins. Radioprotectants and antioxidants affected the pool of blood protein Fe3+-transferrin controlling the synthesis of iron-containing ribonucleotide reductase activity in hematopoietic organs, and hence, the iron-dependent stage in DNA synthesis--dNTP synthesis. Activation of protein synthesis in organs by the studied substances increased the pools of Fe3+-transferrin and Cu2+-ceruloplasmin in the blood, which activated dNTP and DNA synthesis. Activated synthesis of dNTP, DNA, and proteins in the organs and increased pools of studied plasma proteins underlay the formation of body resistance to DNA-damaging factors.

Influence of BHT inclusion on post-thaw attributes of human semen.

The aim of this study was to determine the effect of butylated hydroxytoluene (BHT) supplemented cryopreservation medium on sperm parameters during the freeze-thaw process. In addition, sperm lipid peroxidation, DNA damage, and the amount of reactive oxygen species (ROS) were determined. Semen samples were obtained from 75 donors. Fifteen semen samples were used for optimizing BHT concentration and incubation time and 60 samples were used for the final experiments. After the determination of basic parameters, groups of three sample with similar parameters were pooled and processed by Pure Sperm gradient centrifugation. The semen samples were then diluted with normal freezing medium (control) or a medium containing 0.5 mM BHT (test) for 5 minute and stored in liquid nitrogen. Frozen cryovials were thawed individually for 20 seconds in a water bath (37°C) for evaluation. Freezing extenders supplemented with 0.5 mM BHT led to higher sperm motility and viability compared with control samples (p < 0.001). Furthermore, the addition of BHT decreased malondialdehyde (MDA) formation, DNA fragmentation, and ROS content compared with controls (p < 0.001). Our results showed that the addition of BHT to the freezing medium could be of advantage in reducing ROS and preventing the detrimental effect of ROS on the human sperm function.

Rejuvenescence and extension of an urochordate life span following a single, acute administration of an anti-oxidant, butylated hydroxytoluene.

Two commonly accepted metabolic theories of aging interpret senescence either in terms of the rate of living, where a fixed total metabolic potential is consumed over an expected lifetime (after which the organism wears out and dies) or, in terms of accumulative oxidative damage resulting in progressive and irreversible changes in metabolic pathways. Protocols based on restricted diets, chronically administered anti-oxidants and the use of established lines of organisms resistant to free radical damage support the metabolic theories of aging by revealing, in many cases, significant extensions of life spans or dramatic anti-aging effects. To test the universality of these metabolic hypotheses of aging, we acutely treated ramets (clonal replicates) from old, long-lived colonies of the urochordate Botryllus schlosseri with lethal doses of the anti-oxidant butylated hydroxytoluene (BHT). This group of organisms has a weekly cyclical and highly synchronized developmental process (blastogenesis), during which all existing zooids are removed by massive apoptosis and phagocytosis processes. In animals treated with BHT, blastogenesis was completely arrested and colonies deteriorated to a morphologically chaotic state. Rescued ramets resorbed BHT treated zooids, regenerated entirely new sets of zooids and then revealed: (1) rejuvenescence and enhanced growth rates and in many cases, (2) up to 4.6 times extension of post-treatment life expectancy. Both metabolic theories for senescence were therefore falsified in B. schlosseri. The possible existence of an aging clock that can be set by the environment is suggested.

Dietary butylated hydroxytoluene counteracts with paraquat to reduce the rate of hepatic DNA single strand breaks in senescence-accelerated mice.

The present study was designed to assess the age-related changes of DNA single strand breaks (SSB) in the liver of senescence-resistant and senescence-prone mice, SAMR1/Fky and SAMP1@Fky. In the first series of experiments, the animals were fed a commercial diet for 12-18 months. The mice were killed and the livers were excised at 3- or 6-month intervals for the analysis of the rate of DNA SSB by the ethidium bromide fluorescence method. With advancing age, the rate of DNA SSB was increased in both strains of mice but the increase was significantly higher in SAMP1@Fky than in SAMR1/Fky. In the second series of experiments, the mice were fed one of the following diets for 12 weeks: 20% casein diet (basal diet), basal diet with 300 ppm butylated hydroxytoluene (BHT) added, basal diet with 200 ppm paraquat (PQ) added, and basal diet with 200 ppm PQ plus 300 ppm BHT added. Added dietary PQ increased the rate of DNA SSB in both SAMP1@Fky and SAMR1/Fky. The increases were offset by co-administration of BHT. Dietary BHT, therefore, may suppress the oxidative stress developed by paraquat administration.

Photoprotective effect of vitamins A and E on polyamine and oxygenated free radical metabolism in hairless mouse epidermis.

The purpose of this study was to confirm the photoprotective effect on skin of vitamins A and E, due to inhibition of polyamine synthesis and production of free radicals. These variables were measured in the lumbar epidermis of the female hairless mouse subjected to UVA + B irradiation. Polyamines were assayed in epidermal homogenate by HPLC, and production of oxygenated free radicals was determined by spectrofluorometric assay of malonyl dialdehyde. It was determined that butyl-hydroxy-toluene and vitamin E inhibited production of free radicals (56% and 60%, respectively) and caused a significant reduction in polyamine biosynthesis (P less than 0.01), whereas the inhibitory effect of malonyl dialdehyde induced by vitamin A (30%) had no associated effect on polyamine metabolism.