Choleresis and increased biliary efflux of glutathione induced by phenolic antioxidants in rats

The mechanism by which high doses of the synthetic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) raise hepatic glutathione levels above physiological values was investigated in rats. A single dose of an antioxidant (200 mg/kg; p.o.) reduced the hepatic glutathione content by 17% (BHA) or 36% (BHT) after 5 h, but in contrast levels of 55% (BHA) or 34% (BHT) above controls (7.1 +/- 0.5 mumol GSH-equivalents/g liver wt) were measured 48 h after dosing. Both antioxidants increased basal bile flow (1.37 +/- 0.11 microliter/min per g liver wt) and biliary efflux of total glutathione, i.e. GSH and GSSG, (4.18 +/- 0.97 nmol GSH-eq./min per g) severalfold (up to 250%) over controls 24 h after in vivo antioxidant treatment. The sinusoidal efflux of reduced glutathione (14.9 +/- 2.2 nmol GSH-eq./min per g) was significantly reduced (BHA: 23%; BHT: 41%). The increased glutathione excretion into bile is likely to be independent of the induction of the choleresis. The secretion of bile salts was unaffected by BHA treatment and only temporarily reduced by BHT. Conclusion: phenolic antioxidants increase the hepatic turnover of glutathione by stimulating the biliary efflux of GSH. The resulting shift from a predominantly sinusoidal efflux of GSH in controls (hepato-renal circulation) to a predominantly biliary efflux of GSH in antioxidant-treated animals (entero-hepatic circulation) may lead to increased concentrations of cysteine, glycine and glutamic acid in the portal vein and consequently may stimulate the biosynthesis of GSH by enhanced substrate availability in the liver.     

In vitro genotoxic and cytotoxic effects of some paraben esters on human peripheral lymphocytes

Parabens (PBs) are p-hydroxybenzoic acid ester compounds commonly employed as antimicrobial preservatives, mainly in food, cosmetic, and pharmaceutical products. The aim of the present study was to investigate the genotoxic and cytotoxic effects of some paraben esters (butyl paraben, propyl paraben, isobutyl paraben, and isopropyl paraben) on human peripheral lymphocytes, using in vitro sister chromatid exchange (SCE), chromosome aberration (CA), and cytokinesis-block micronucleus (CBMN) tests. Lymphocyte cultures were treated with four concentrations of PBs (100, 50, 25 and 10?µg/mL) for 24 and 48?h. Paraben esters significantly induced MN formations as compared to solvent control. Furthermore, butyl paraben and propyl paraben increased MN formations a concentration-dependent manner at 24 and 48?h. PBs increased the CA at 24 and 48?h. However, this increase was not meaningful for butyl paraben and isopropyl paraben at 48?h when compared with solvent control. Butyl, isobutyl, and isopropyl paraben significantly increased the SCE at 24 and 48?h. However, propyl paraben did not induce SCE meaningfully in both treatment periods. A significant decrease in the cytokinesis-block proliferation index and mitotic index was observed in cells exposed to all concentrations of PBs at 24 and 48?h. However, proliferation index was not affected at all concentrations of PBs after 24?h treatment, although it was decreased at the highest concentration of PBs at 48?h. It is concluded that all of the paraben esters used in this study have highly genotoxic and cytotoxic effects on human lymphocytes cells in vitro.     

Intake of phenolic antioxidants from foods in Canada

The potential dietary intake of butylated hydroxyanisole (BHA) in Canada was estimated using dietary recall data on food consumption and maximum permitted use levels for this antioxidant. Based on these estimates, it is concluded that the dietary intake of BHA and other permitted phenolic antioxidants (BHT and propyl gallate) is unlikely to exceed 1 mg/kg body weight/day and on average is less than 0.4 mg/kg body weight/day. Canadian dietary intake estimates compare favourably with those of the United States.     

Effect of four synthetic antioxidants on the formation of ethylene from methional in rat liver microsomes

Four commonly used food antioxidants, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate and octyl gallate, were tested for their ability to inhibit the formation of ethylene from methional in NADPH-oxidizing rat liver microsomes. It is assumed that the action of the antioxidants on ethylene formation reflects their free radical scavenging activity. Only propyl gallate and octyl gallate are efficient inhibitors of ethylene formation. BHT is inhibitory only at very high concentrations, and BHA tends to increase ethylene formation. It is concluded that gallic acid ester antioxidants may possess a protective potential during chemical-induced microsomal oxidations.     

Variation in the phenolic content and in vitro antioxidant activity of Sorbus aucuparia leaf extracts during vegetation

Seasonal variation in the antioxidant activity and content of phenolic compounds was studied for the 70% methanol extracts of Sorbus aucuparia leaves harvested monthly over the full course of the growing season. The antioxidant potential of the extracts was evaluated using two complementary in vitro tests: the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assay and the AAPH [2,2'-azobis-(2-amidinopropane)dihydrochloride]-induced [corrected] linoleic acid (LA) peroxidation test. The radical-scavenging capacities of the extracts towards the DPPH radical were in the range of 0.40 to 0.57 millimolar Trolox equivalents/g dry weight of the leaves. They were significantly correlated (r = -0.8480, p < 0.05) with the results of the LA-peroxidation test, indicating the S. aucuparia leaf extracts to be universal antioxidants. Significant linear correlations were also found between the different antioxidant potentials and total phenolic contents as estimated by the Folin-Ciocalteu method and further verified by serial determinations of proanthocyanidins, chlorogenic acid isomers and flavonoids ([r] in the range of 0.81-0.97, p < 0.05). As the best antioxidant capacities and the highest phenolic contents were found for the leaf samples harvested during the three summer months (June, July and August), this period could be considered to be optimal for cost-effective production of natural health products. For the leaf samples collected in July, the values of EC50 and IC50 for the two antioxidant tests were 2.02 and 93.45 μg [corrected] phenolics/mL, respectively. These antioxidant capacities were found to be higher or comparable to those of synthetic and natural phenolic antioxidants, such as BHT (butylated hydroxytoluene), BHA (butylated hydroxyanisole), TBHQ (tert-butylhydroquinone), quercetin and Trolox.     

The toxicological implications of the interaction of butylated hydroxytoluene with other antioxidants and phenolic chemicals

Butylated hydroxyanisole (BHA) enhanced both the in vitro peroxidase-catalysed covalent binding of butylated hydroxytoluene (BHT) to microsomal protein and the formation of BHT-quinone methide. Eugenol, methylparaben, vanillin, guaiacol, ferulic acid and several other phenolic compounds commonly used in food and cosmetic products also enhanced the metabolic activation of BHT. BHA was the most effective compound tested. Microsomes from lung, bladder, kidney medulla and small intestine of various animal species, including man, were also able to support this interaction of BHA and BHT using either hydrogen peroxide or arachidonic acid as the substrate. These in vitro observations were extended to an in vivo mouse lung model. Subcutaneous injections of BHA significantly enhanced the lung/body weight ratio of mice given intraperitoneal injections of subthreshold doses of BHT. The toxicological implications of the interactions of BHT with other antioxidants and phenolic chemicals and their potential relevance to human risk are discussed.     

Antioxidant and Free Radical Scavenging Effects of Lippia nodiflora

This study evaluated the in vitro antioxidant potential of methanol extract of Lippia nodiflora Mich. (Verbenaceae) (MELN). The different antioxidants assays, including total antioxidant activity, reducing power, free radical, superoxide anion radical, hydroxyl radical, hydrogen peroxide, nitric oxide scavenging, and total phenolic content, were studied. MELN exhibited potent total antioxidant activity that increased with increasing amount of extract concentration (50, 100, 200, and 400 μg/mL), which were compared with standard drug α -tocopherol (400 μg/mL). The different concentrations of MELN and α -tocopherol showed inhibition of 49.07%, 58.96%, 63.07%, 68.29%, and 74.59%, respectively, on peroxidation in linoleic acid emulsion. In addition, MELN had effective reducing power, free radical scavenging, superoxide anion radical scavenging, hydroxyl radical scavenging, hydrogen peroxide radical scavenging, and nitric oxide scavenging activity, and total phenolic content depending on concentration. These various antioxidant activities were compared with standard antioxidants such as BHA, BHT, catechin, and α -tocopherol.     

Estimate of the maximal daily dietary intake of butylated hydroxyanisole and butylated hydroxytoluene in The Netherlands

The daily dietary intake of the phenolic antioxidants butylated hydroxyanisole (BHA) and/or butylated hydroxytoluene (BHT) was estimated using data obtained from a nationwide dietary record survey carried out in The Netherlands in 1987/1988. The estimates were based on the fat content of selected food categories and their respective maximum permitted levels of BHA and/or BHT. The results indicate that it is unlikely that the current acceptable daily intake for BHA (0-0.05 mg/kg body weight) is surpassed, even in individuals with an extremely high caloric intake, except in extreme cases in 1-6-year-olds. However, it cannot be excluded that the acceptable daily intake for BHT (FAO/WHO: 0-0.125 mg/kg; EEC: 0-0.05 mg/kg) is exceeded in all age and sex groups, but particularly in children aged 1-6 years.     

An examination of the thermodynamics of fusion, vaporization, and sublimation of several parabens by correlation gas chromatography

The vaporization, fusion, and sublimation enthalpies of methyl, ethyl, propyl, and butyl paraben are reported and compared with literature values. The vaporization enthalpies were measured by correlation gas chromatography and the fusion enthalpies by differential scanning calorimetry. Adjusted to T = 298.15 K, these enthalpies were combined to yield the sublimation enthalpy. The results compare favorably to some of the literature values but do not support the reversal in magnitude of both the vaporization and sublimation enthalpy previously reported for propyl and butyl paraben. The following fusion and vaporization enthalpies were measured for methyl through to butyl paraben, respectively: ΔH(fus) (T(fus) ) 26.3 ± 0.1 (398.6 K), 26.5 ± 0.1 (388.5 K), 27.3 ± 0.1 (368.8), and 25.9 ± 0.3 (340.7 K) kJ·mol(-1); ΔH(vap) (298.15 K) 79.5 ± 0.5, 84.0 ± 0.5, 89.7 ± 0.6, and 95.8 ± 0.6 kJ·mol(-1). The results are believed to be accurate to ± 4 kJ·mol(-1).     

A study of parabens and bisphenol A in surface water and fish brain tissue from the Greater Pittsburgh Area

Pollution from xenoestrogens has been discovered in the aquatic environment of the Greater Pittsburgh Area and is suspected to be caused by the failing sewer system. Personal care products and plasticizers have the potential to enter the water supply though treated and untreated sewage. Many of these compounds are suspected xenoestrogens. Paraben detection in surface waters was as follows: methyl paraben ranged between 2.2 to 17.3 ppt; ethyl paraben was not detectable; propyl paraben was detected at 9.2 and 12.0 ppt; butyl paraben was detected at 0.2 ppt. BPA was detected between 0.6 and 15.4 ppt. Estrogenic potential of extracts from fish brain tissue was tested via Bromodeoxyuridine MCF-7 analysis and paired with HPLC–MS to investigate the presence of xenoestrogens. All samples were non-detectable for parabens. BPA was detected in 44 of the 58 samples, with a range from non-detectable to 120 pg/g. BCFs were calculated. Results were statistically significant for location of capture (p < 0.05) and correlation existed between estrogenicity and BPA.     

Determination of warfarin in human plasma by high performance liquid chromatography

A simple procedure for the determination of warfarin in plasma samples is described. The assay involves a single ethereal extraction of warfarin and its internal standard, propyl paraben, from an acidified sample. The extract is evaporated to dryness at 45 degrees C and the residue is redissolved in methanol (50 microliters). A 25-microliters aliquot is injected into the liquid chromatograph and eluted with acetonitrile - 10 mM phosphate buffer at pH 3.5 (48:52, v/v) on a C8 pre-column linked to a 5-microns C8 reversed-phase column. The flow rate was set at 1.5 ml min-1 and UV detector at 215 nm. The method has been used to determine plasma concentrations of warfarin in Chinese patients with recent atrial fibrillation scheduled for electrocardioversion.     

Compatibility of sennoside A and B with pharmaceutical excipients

This study reports the incompatibility of sennoside A and B with the following commonly used pharmaceutical excipients: stearic acid, sodium carbonate, glucose, lactose, propyl paraben, sodium carbonate, stearic acid, citric acid, PEG, and sorbitol. Drug-excipient compatibility was tested using thermal (DSC) and analytical (HPLC) methods of analysis. Compatibility evaluation showed that dry powder mixtures could be used to formulate sennoside A and B products. However, when mixed with water--propyl paraben, sodium carbonate, stearic acid, citric acid, PEG, and sugar derivatives such as lactose, glucose and sorbitol--should not be used in sennoside containing products.     

Effect of antioxidants and silicates on peroxides in povidone

Reactive peroxides in povidone often lead to degradation of oxidation-labile drugs. To reduce peroxide concentration in povidone, the roles of storage conditions, antioxidants, and silicates were investigated. Povidone alone and its physical mixtures with ascorbic acid, propyl gallate, sodium sulfite, butylated hydroxyanisole (BHA), or butylated hydroxytoluene (BHT) were stored at 25 °C and 40 °C, at 11%, 32%, and 50% relative humidity. In addition, povidone solution in methanol was equilibrated with silicates (silica gel and molecular sieves), followed by solvent evaporation to recover povidone powder. Peroxide concentrations in povidone were measured. The concentration of peroxides in povidone increased under very-low-humidity storage conditions. Among the antioxidants, ascorbic acid, propyl gallate, and sodium sulfite reduced the peroxide concentration in povidone, whereas BHA and BHT did not. Water solubility appeared to determine the effectiveness of antioxidants. Also, some silicates significantly reduced peroxide concentration in povidone without affecting its functionality as a tablet binder. Porosity of silicates was critical to their ability to reduce the peroxide concentration in povidone. A combination of these approaches can reduce the initial peroxide concentration in povidone and minimize peroxide growth under routine storage conditions.     

Partitioning of parabens between phases of submicron emulsions stabilized with egg lecithin

Partitioning of methyl and propyl parabens (methyl and propyl hydroxybenzoate, paraben M and P) between the major phases in the parenteral submicron emulsions was studied. The investigated emulsions contained 10% or 20% soya-bean oil, 1.2% or 2.4% egg lecithin, 0.18% or 0.36% paraben M and 0.02% or 0.04% paraben P. The aqueous phase was obtained by ultracentrifugation, and subsequently, it was subjected to ultrafiltration, which procedure allowed to distinguish between the fractions of free preservatives (Fw) and incorporated in the liposomal or micellar region (Flm). The fractions present in the oily phase and in the interface were calculated. Depending on the formulation, Fw was 17-31% and 2.3-6.0% for paraben M and P, respectively. The Flm values were in a very narrow range, i.e. 3.0-6.0% for both preservatives. Substantial accumulation, i.e. 38-58% was found in the interface and the partitioning into this region was related to the oil/lecithin ratio rather than to lipophilicity of the preservative.     

Gastric retention and delayed absorption of a large dose of butylated hydroxytoluene in the rat

1. After a single oral dose of 800 mg/kg of butylated hydroxytoluene to rats, the plasma concentration of 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT quinone methide), an active metabolite of BHT, reached a maximum 18 h after dosing. 2. The gastrointestinal content of BHT remained constant from 0.5 to 12 h, and began to decline 18 h after dosing. 3. BHT concentrations in epididymal and subcutaneous adipose tissues also attained maxima 18 h after dosing. 4. Volumes and weights of stomachs and contents, and volumes of gastric contents of rats given 800 mg/kg BHT, were 2-3 times larger than those of controls 4-7 h after dosing while there were no significant differences at 24-27 h after dosing. 5. Retention of ingested material in the stomachs was also observed when 18-h starved rats were given 800 mg/kg BHT. 6. Thirty minutes after intraduodenal administration of 800 mg/kg BHT to anaesthetized rats. BHT, but not BHT quinone methide, was 0.4-1.4 micrograms/ml in portal vein plasma. No BHT was detected in plasma from the aorta descendens. BHT and/or BHT radical, and BHT alone, were also found in liver and epididymal adipose tissue at 7-20 and 25-40 micrograms/g wet weight, respectively. 7. These results indicate that delay in attainment of maximum concentration of BHT or its metabolites in internal organs may be due to the long retention in the stomach, caused by high dosage and the inhibitory effect of BHT on gastric function. Moreover, most BHT may not enter lymphatic fluid but it can be absorbed into portal blood.     

Safety assessment of propyl paraben: a review of the published literature.

Propyl paraben (CAS no. 94-13-3) is a stable, non-volatile compound used as an antimicrobial preservative in foods, drugs and cosmetics for over 50 years. It is an ester of p-hydroxybenzoate. Propyl paraben is readily absorbed via the gastrointestinal tract and dermis. It is hydrolyzed to p-hydroxybenzoic acid, conjugated and the conjugates are rapidly excreted in the urine. There is no evidence of accumulation. Acute toxicity studies in animals indicate that propyl paraben is relatively non-toxic by both oral and parenteral routes, although it is mildly irritating to the skin. Following chronic administration, no-observed-effect levels (NOEL) as high as 1200-4000 mg/kg have been reported and a no-observed-adverse-effect level (NOAEL) in the rat of 5500 mg/kg is posited. Propyl paraben is not carcinogenic, mutagenic or clastogenic. It is not cytogenic in vitro in the absence of carboxyesterase inhibitors. The mechanism of propyl paraben may be linked to mitochondrial failure dependent on induction of membrane permeability transition accompanied by the mitochondrial depolarization and depletion of cellular ATP through uncoupling of oxidative phosphorylation. Sensitization has occurred when medications containing parabens have been applied to damaged or broken skin. Parabens have been implicated in numerous cases of contact sensitivity associated with cutaneous exposure, but high concentrations of 5-15% in patch testing are needed to elicit reaction in susceptible individuals. Allergic reactions to ingested parabens have been reported, although rigorous evidence of the allergenicity of ingested paraben is lacking.     

Studies on antioxidants: their carcinogenic and modifying effects on chemical carcinogenesis

Studies were conducted on the carcinogenic activity of butylated hydroxyanisole (BHA) in rats and hamsters. To obtain information concerning the mechanism of action of BHA on the forestomach, the following areas were examined: the effects of 12 phenolic compounds structurally related to BHA on the hamster forestomach, the effects of combinations of BHA and other antioxidants on the rat forestomach, and the metabolism of BHA in the forestomach. Also examined were the effects of several antioxidants on two-stage carcinogenesis in rats. Squamous-cell carcinomas were induced in the forestomach of rats and hamsters fed BHA. In a limited study, 1 of 13 hamsters developed a squamous-cell carcinoma. The tumorigenic action of crude BHA on the forestomach was largely due to the action of 3-tert-BHA. p-tert-Butylphenol and 2-tert-butyl-4-methylphenol induced pronounced hyperplasia and papillomas in the hamster forestomach. BHA and other antioxidants, particularly propyl gallate and ethoxyquin, showed additive effects in inducing forestomach hyperplasia and cytotoxicity. Neither BHA nor its metabolites were found in the forestomach epithelium, although small amounts of metabolites were detected in the stomach contents. Thus, a direct action on the stomach epithelium may be exerted by BHA itself or by metabolites formed on interaction of BHA with gastric juice. BHA enhanced forestomach carcinogenesis initiated in rats by N-methyl-N'-nitro-N-nitrosoguanidine or N-methylnitrosourea (MNU) and enhanced urinary bladder carcinogenesis initiated by MNU or N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). In contrast, it inhibited carcinogenesis initiated in the liver by either diethylnitrosamine or N-ethyl-N-hydroxyethylnitrosamine (EHEN) and mammary carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene (DMBA). BHT promoted urinary bladder carcinogenesis initiated by BBN or MNU and thyroid carcinogenesis initiated by MNU, but inhibited ear-duct carcinogenesis initiated by DMBA. Ethoxyquin promoted EHEN-initiated kidney carcinogenesis, but inhibited both DMBA-initiated mammary and EHEN-initiated liver carcinogenesis. Sodium ascorbate promoted forestomach and urinary bladder carcinogenesis, and sodium erythorbate also enhanced urinary bladder carcinogenesis. alpha-Tocopherol inhibited ear-duct carcinogenesis. No antioxidants tested had any effect on glandular stomach carcinogenesis. Thus antioxidants have independent modifying (promoting or inhibitory) effects in different organs.     

Effect of alpha-tocopherol and di-butyl-hydroxytoluene (BHT) on UV-A-induced photobinding of 8-methoxypsoralen to Wistar rat epidermal biomacromolecules in vivo

The possible formation of singlet oxygen via photoexcited psoralens has been associated with the occurrence of, amongst others, erythema. Therefore it has been suggested to combine PUVA with the topical or systemic administration of antioxidants. However, the effect of these antioxidants on the photobinding of psoralens to DNA, which is held responsible for the anti-proliferative effect, should be taken into account. In the present study the effect of two phenolic antioxidants, alpha-tocopherol (AT) and butylated hydroxytoluene (BHT), on the in vivo photobinding of 8-methoxypsoralen (8-MOP) to not only epidermal DNA, but also proteins and lipids was determined. After topical application of an ethanolic antioxidant solution onto the shaven skin of Wistar rats, labeled 8-MOP was applied using the same solvent. After this the rats were exposed to UV-A. By separating epidermal lipids, DNA/RNA and proteins by a selective extraction method, irreversible binding of 8-MOP to each of these biomacromolecules was determined. Both AT and BHT caused a decrease in the photobinding of 8-MOP to epidermal DNA and proteins. To investigate the underlying mechanism of this protection, the effect of AT was compared with that of AT-acetate. It also proved helpful to study the effects of the antiooxidants on the photobinding of another photosensitizer, namely chlorpromazine. From these experiments it was concluded that AT and BHT affect 8-MOP photobinding by quenching reactive 8-MOP intermediates, involving the phenolic hydroxyl group of the antioxidants. BHT offered protection against lipid binding of 8-MOP but AT, especially at high concentrations, enhanced the UV-A-induced binding of 8-MOP to lipids. This is noteworthy with respect to the current interest in the role of 8-MOP-lipid adducts in PUVA therapy.     

The antioxidant activity of some curcuminoids and chalcones

The antioxidant properties of the synthetic compound (C1)–(C8), which comprised 7 curcuminoids and a chalcone, were evaluated by two complementary assays, DPPH and β-carotene/linoleic acid. It was found that, in general, the free radical scavenging ability of (C1)–(C8) was concentration-dependent. Compounds (C1) and (C4), which contained (4-OH) phenolic groups, were found to be highly potent antioxidants with higher antioxidant values than BHT suggesting that synthetic curcuminoids are more potent antioxidants than standard antioxidants like BHT. Using β-carotene-linoleic acid assay, only the water-soluble 2, 4,6-trihydroxyphenolic chalcone (C5) showed 85.2 % inhibition of the formation of conjugated dienes reflecting on its potent antioxidant activity.     

Gas chromatographic determination of dimethophrine in biological specimens of rabbit.

A method to measure the alpha-sympathomimetic amine, 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-monomethyl-amino-ethanol (dimethophrine, DMP), was developed and applied for the determination of the drug in biological specimens. The gas chromatographic degradation of the drug was investigated, and in order to avoid this process, DMP is converted to the trifluoroacetyl derivative and assayed by gas liquid chromatography on an OV 1' column. The minimum detectable amount is 25 pg per injection. Linearity (using hexachlorobenzene as internal standard for quantitation) was found up to mug amounts of the substance, without any interference with endogenous substrates. Values from plasma, red blood cells and urine in rabbits treated with a single dose of DMP (10 mg/kg) are also reported.