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.     

Induction of apoptosis by mono(2-ethylhexyl)phthalate (MEHP) in U937 cells

Treatment of U937 cells with mono(2-ethylhexyl)phthalate (MEHP) for 20 h led to a dose-dependent loss of cell viability, assessed by propidium iodide (PI) staining with fluorescent activated cell sorting (FACS) analysis. The cytotoxic behavior of MEHP is attributed to the induction of apoptosis. MEHP induced activation of caspase-3, internucleosomal DNA fragmentation and the morphological features of nuclear apoptosis. Analysis with LightCycler quantitative RT-PCR demonstrated the decrease of bcl-2 and increase of bax mRNA levels. Peroxisome proliferator-activated receptor (PPAR) gamma antagonists, bisphenol A diglycidyl ether (BADGE) and GW9662, significantly inhibited the MEHP-induced caspase-3 activity and apoptotic nuclear morphological changes. Furthermore, a PPARgamma ligand, rosiglitazone synergized the MEHP-induced caspase-3 activity. These results suggest that MEHP can induce apoptosis in U937 cells through modulation of the balance of bcl-2/bax in part by PPARgamma activation.     

Studies on estrogenic activities of food additives with human breast cancer MCF-7 cells and mechanism of estrogenicity by BHA and OPP

Estrogenic activities of more than 90 chemicals including food additives, foodstuffs of plant origin, and some chemicals, which could be orally ingested, were examined by assaying estrogen receptor (ER)-dependent proliferation of MCF-7 cells. Among 66 food additives, 17 compounds stimulated the proliferation, but their concentrations giving maximal cell yield were higher than that of 17 beta-estradiol and their estrogenic activities were weak. Flavonoids had relatively strong estrogenic activities. In the assay of ER competitive binding to human ER alpha and ER beta in vitro, the antioxidant t-butylhydroxyanisole (BHA) had the capacity to compete with 17 beta-estradiol, while the capacity of o-phenyl phenol (OPP) was too small to calculate. Both BHA and OPP induced a decrease in gene expression of ER alpha and an increase in that of progesterone receptor in a time-dependent manner. These effects were similar to that of 17 beta-estradiol, a though much higher concentrations were required for these compounds than 17 beta-estradiol. These results may suggest that we should be careful not to ingest excessive food additives.     

Molecular identification of human Diphyllobothrium nihonkaiense using mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequence

Identification of Diphyllobothrium species has been carried out based on their morphology, especially sexual organs. In addition to these criteria, PCR-based identification methods have been developed recently. A 20 year-old Japanese living in Kochi Prefecture passed tapeworm. He was successfully treated with single dose of gastrografin. We examined the morphologic features of the proglottids and eggs using histology and scanning electron microscope. We also analyzed mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the proglottids. The causative tapeworm species was identified as D. nihonkaiense based on the results of morphologic features and genetic analysis. We discussed the advantage of PCR-based identification methods of Diphyllobothrium species using cox1 sequence in the clinical laboratory.     

Formation of 8-hydroxydeoxyguanosine in calf thymus DNA treated in vitro with phenylhydroquinone, the major metabolite of O-phenylphenol

The generation of 8-hydroxydeoxyguanosine (8OHdG) in calf thymusDNA treated with O-phenylphenol (OPP) or its major metabolites,phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), was studied.The content of 8OHdG residues was increased in DNA treated withPHQ, and the generation of 8OHdG was highly dependent on PHQconcentration. PBQ had little effect on the formation of 8OHdG,and OPP had no effect. The formation of 8OHdG by PHQ was reducedby oxygen radical scavengers such as catalase, sodium benzoateand sodium azide. The PHQ-induced 8OHdG formation was acceleratedby the addition of CuCI or CuCI₂ to the reaction mixture, butwas decreased by the addition of chelating agents such as EDTA,bathocuproinedisulfonic acid disodium salt (bathocuproine disulfonate)and O-phenanthroline. These results demonstrate that hydroxylradicals generated in the process of oxidation of PHQ contributeto the formation of 8OHdG in DNA, and copper ions facilitatethe oxidative DNA damage. Copper ions greatly accelerated thePHQ-induced DNA cleavage in vitro, although they had no effecton cleavage without PHQ. On the other hand, DNA cleavage occurredby the addition of FeCl₂ in the absence and presence of PHQ.FeCI₂ stimulates 8OHdG formation only slightly with or withoutPHQ. Furthermore, the stimulatory effect of FeCl₂ on 8OHdG formationwas observed even in the presence of EDTA. The formation of8OHdG in bladder DNA is likely to be one of a series of eventsleading to bladder tumors seen in rats fed OPP-containing diet.     

Estimation of estrogenic and anti-estrogenic activities of some phthalate diesters and monoesters by MCF-7 cell proliferation assay in vitro

Phthalates are man-made chemicals abundantly found in the environment. Estrogenic activities of phthalate di and monoesters were studied by in vitro assay of human breast cancer MCF-7 cell proliferation. Since phthalate monoesters are formed from diesters by degradation and are found in the environment, we selected some phthalate monoesters in addition to diesters. Among 19 compounds tested, dicyclohexyl phthalate (DCHP), di(2-ethylhexyl) phthalate (DEHP) and butyl benzyl phthalate (BBP) were found to have estrogenic activities, all of which were completely suppressed by the addition of pure anti-estrogen ICI 182780. DCHP stimulated cell proliferation with maximal cell yield at 5 x 10(-5) M. Its estrogenic potency was approximately 1700000 times less than that of 17beta-estradiol. DEHP and BBP stimulated cell proliferation only slightly at >10(-3) M. No other phthalate diesters or monoesters tested were estrogenic. Anti-estrogenic activities were also examined by estimating the suppression of cell proliferation in the presence of 10(-11) M 17beta-estradiol. Mono-n-pentyl phthalate (MPP), monocyclohexyl phthalate (MCHP), monobenzyl phthalate (MBZP), monoisopropyl phthalate (MIPrP) and BBP were suggested to have anti-estrogenic activities at higher than 10(-4) M. Among commonly used phthalate esters and those with related structures, some were found to be estrogenic and others were anti-estrogenic in vitro.     

DNA cleavage by metabolites of butylated hydroxytoluene

The effect of butylated hydroxytoluene (BHT) and its metabolites on DNA cleavage in vitro was studied with supercoiled plasmid DNA, pUC18, by agarose gel electrophoresis. Among several BHT metabolites, 2,6-di -t-butyl-p-benzoquinone (BHT-quinone) caused cleavage of supercoiled DNA (form I) at a concentration as low as 1 × 10^–6 M. The relative amount of linear form (form III) was increased with increasing concentration of BHT-quinone. 2,6-Di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHT-peroxyquinol) and 3,5-di-t-butyl-4-hydroxybenzaldehyde (BHT-CHO) also cleaved DNA, but to a lesser extent than BHT-quinone. No DNA cleavage was detected by BHT, 2,6-di-t-butyl-4-hydroxymethyl phenol (BHT-OH), 3,5-di-t-butyl-4-hydroxybenzoic acid (BHT-COOH), 2,6-di-t-butyl-4-hydroxy-4-methyl-2,5-cyclohexadienone (BHT-quinol) or 2,6-di-t-butyl-4-methylene-2,5-cyclohexadienone (BHT-quinone methide). The DNA cleavage by BHT-quinone was inhibited by oxygen radical scavengers including Superoxide dismutase (SOD), catalase, polyethylene glycol,t-butyl alcohol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine, while it was enhanced by the addition of FeCl₂. The production of Superoxide radical in a solution of BHT-quinone was confirmed by cytochrome c reduction assay. Superoxide was not produced by BHT or other BHT metabolites except for BHT-quinone. These results suggest that BHT-quinone, one of the principal metabolites of BHT, cleaves DNA strands via its generation of oxygen radicals. Such modification of DNA observed in vitro may be relevant to genotoxicity by BHT after metabolic activation in vivo.