Propyl gallate exposure affects the mouse 2-cell stage embryonic development through inducing oxidative stress and autophagy.

Propyl gallate (PG), owing to its exceptional antioxidant properties, is extensively used in industries such as food processing. The potential harmful impacts of PG have sparked concern among people. It has been reported that exposure of PG has certain reproductive toxicity, which can affect the maturation of mouse oocytes and induce testicular dysfunction. However, its impact on early embryonic development is still unclear. In this study, we explored the toxic effects and potential mechanisms of PG on mouse 2-cell stage embryonic development. The results showed that exposure of PG can decrease the development of 2-cell stage embryos and repress the development of 4-cell stage embryos. Further study found that PG could induce intracellular oxidative stress and the accumulation of DNA damage in 2-cell stage embryos. Moreover, exposure of PG impaired the function of mitochondria and lysosomes in 2-cell stage embryos, thereby triggering the occurrence of autophagy. In addition, exposure of PG altered the epigenetic modification of 2-cell stage embryos, displaying a decreased level of DNA methylation and an increased level of H3K4me3. In summary, our results indicated that exposure of PG can damage the development of mouse 2-cell stage embryos by inducing oxidative stress, DNA damage, and autophagy, and altering epigenetic modification.

Pharmacokinetic and toxicological overview of propyl gallate food additive.

The progressive use of food additives in "ultra-processed" food has increased attention to them. Propyl gallate (PG) is an essential synthetic preservative that commonly used in food, cosmetics, and pharmacies as an antioxidant. This study aimed to outline the existing evidence on the toxicological studies of PG including its physicochemical properties, metabolism, and pharmacokinetics effects. The methods include updated searches for the relevant databases. The EFSA has evaluated the use of PG in food industry. It establishes an acceptable daily intake (ADI) of 0.5 mg/kg bw per day. Based on exposure assessment, it can be concluded that at the current level of use, PG is not of safety concern.

Synthetic phenolic antioxidant propyl gallate induces male infertility through disruption of calcium homeostasis and mitochondrial function.

Propyl gallate (propyl 3,4,5-trihydroxybenzoate, PG) is a phenolic antioxidant that has been used for oil-containing foods to prevent acidification. Owing to its antioxidant properties, PG has been applied to various fields and active research is currently underway to prove PG as an anticancer agent. However, there are still concerns about PG as a possible reproductive toxicant. Therefore, we determined whether PG induced male infertility. Our results indicated that PG induced testicular dysfunction in both Leydig and Sertoli cells via suppression of cell viability and steroidogenesis. These normal testis functions were destroyed by PG-induced mitochondrial dysfunction and calcium homeostasis dysregulation. In addition, PG disrupted the expression of several genes associated with the testis function and induced endoplasmic reticulum stress. Furthermore, we verified PG-induced mRNA expression changes in steroidogenesis enzymes and hormone receptors in vitro and in vivo. From the results of the qPCR analysis, we further confirmed the PG-mediated reduction in the mRNA expression of genes related to testis functions by in situ hybridization. Finally, we demonstrated that PG induced testicular toxicity via the disruption of mitochondrial or ER function and the inhibition of testicular development-related genes in mice.

Estrogenic and anti-estrogenic activity of butylparaben, butylated hydroxyanisole, butylated hydroxytoluene and propyl gallate and their binary mixtures on two estrogen responsive cell lines (T47D-Kbluc, MCF-7).

The estrogenic and anti-estrogenic effects of butylparaben (BuPB), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) were evaluated for individual compounds as well as for binary mixtures, using an estrogen-dependent reporter gene assay in T47D-Kbluc breast cancer cells and an estrogen-dependent proliferation assay in MCF-7 breast cancer cells. In terms of estrogenicity the potency of the selected compounds increased from BHA < PG < BuPB in the luciferase assay (with BHT showing no significant estrogenic activity), while in the proliferation assay the following order was observed: BHT < BHA < BuPB (with PG showing no significant estrogenic activity). Non-monotonic dose-response curves were obtained for BuPB (in both assays) and PG (in the luciferase assay), respectively. In the presence of estradiol, a significant anti-estrogenic activity was observed in both cell lines for PG, BuPB and BHA, while BHT showed weak anti-estrogenic activity only in T47D-Kbluc cells. The evaluation of binary mixtures confirmed the endocrine disruptive potential of the compounds, their individual potency being correlated with that of the mixtures. All mixtures were able to reduce the estradiol-induced luminescence or cell proliferation, an effect that was accurately predicted by the dose addition mathematical model, suggesting the same (or at least partially overlapping) modes of action for the tested compounds. The results of the present study emphasize the importance of a cumulative risk assessment of endocrine disruptors.

Determination of developmental toxicity of zebrafish exposed to propyl gallate dosed lower than ADI (Acceptable Daily Intake).

Propyl gallate (PG) is an antioxidant substance widely used in cosmetics, pharmaceutical and food industries. The aim of this study was to evaluate the potential toxic effect of PG injected to zebrafish embryos. To this end, zebrafish embryos were exposed to PG with 0, 1, 10 and 50 ppm concentrations which are lower than ADI and were monitored at 24, 48, 72 and 96 hpf. Survival rate, hatching rate and malformations were evaluated during this period. Moreover, it has been detected the accumulation of fluorescence signal of ROS and apoptotic cell in whole body at the end of 96 hpf. According to results, survival rate slightly decreased in highest concentration, and PG accelerated hatching in 1 and 10 ppm concentrations whereas delayed in 50 ppm concentration. In addition, it has been detected accumulation of fluorescence signal of ROS and apoptotic cells in a dose dependent-manner. Consequently, it has been considered that increased embryonic or larval malformation in this study may have been caused by ROS-induced apoptosis. The obtained data suggested that the developmental toxicity caused by PG and/or multiple hydroxyl groups arose when PG hydrolyze to gallic acid is probably triggered by the induction of ROS formation and consequent apoptosis.

Propyl gallate induces cell death and inhibits invasion of human trophoblasts by blocking the AKT and mitogen-activated protein kinase pathways.

Propyl gallate (PG) is an antioxidant widely used in food additives, cosmetics, adhesives, and lubricants. PG protects the oils in food products from reacting with hydrogen peroxide and oxygen free radicals, thus preventing spoilage. It is known to have both protective and cytotoxic effects on various cells, but its effects on human trophoblasts remain unclear. Therefore, we investigated the effects of PG on proliferation, apoptosis, and invasiveness of human trophoblasts using an immortalized HTR8/SVneo cell line. We found that PG significantly reduced proliferation of and induced apoptosis in HTR8/SVneo cells. In addition, the invasiveness of HTR8/SVneo cells was attenuated in response to PG. Signaling pathways including the PI3K/AKT and MAPK pathways involved in the proliferation and invasiveness of human trophoblasts were regulated by PG treatment in HTR8/SVneo cells. We confirmed that mitochondrial membrane disruption and Ca2+ overload were markedly elevated in response to PG treatment, suggesting that PG-induced apoptosis is closely related to mitochondrial dysfunction and further PG-induced apoptosis in HTR8/SVneo cells is related to endoplasmic reticulum (ER) stress. Collectively, these results indicate that PG exerts harmful effects on human trophoblasts; therefore, exposure to PG in early pregnancy is predicted to cause abnormal implantation and placental development.

Immunofluorescence evaluation of 4-hydroxynonenal and 8-hydroxy-2-deoxyguanosine activation in zebrafish (Daino rerio) larvae brain exposed (microinjected) to propyl gallate.

Propyl gallate (PG) is a chemical compound obtained by esterification of propanol with gallic acid. Due to its antioxidative properties, it is widely used in cosmetics and pharmaceutical industries as well as to protect the oils in foods such as butter, milk-based desserts, chewing gum, mayonnaise, meat, soups, cereals, spices and seasonings from rancidity. This study has been designed to assessment 8-OHdG and 4-HNE activity, and histopathological changes in the brain tissues of zebrafish larvae, which is a lecithotrophic organism, after 96 h of PG exposure via microinjecting to yolk sac of embryo. To this end, approximately 5 nL of various concentrations of PG (1, 10, and 50 ppm) has been injected into yolk sac of fertilized embryo (final exposure concentrations are 5, 50, 250 pg/egg) with micro manipulator system. After 96 h exposure time, propyl gallate caused immunofluorescence positivity of 8-OHdG and 4-HNE in the brain tissues of zebrafish larvae. PG was not effect brain tissue histopathological in low concentrations (1 and 10 ppm) but highest concentration (50 ppm) caused degenerative changes in brain. These results suggests that PG treatment could lead oxidative DNA damage by causing an increase 8-OHdG and 4-HNE activities. This strategy will enable us to better understand the mechanisms of propyl gallate in brain tissues of zebrafish larvae.

Disruption of DNA Damage-Response by Propyl Gallate and 9-Aminoacridine.

The DNA-damage response (DDR) protects the genome from various types of endogenous and exogenous DNA damage, and can itself be a target of certain chemicals that give rise to chromosomal aberrations. Here, we developed a screening method to detect inhibition of Mediator of DNA damage Checkpoint 1 (MDC1) foci formation (the Enhanced Green Fluorescent Protein (EGFP)-MDC1 foci formation-inhibition assay) using EGFP-MDC1-expressing human cells. The assay identified propyl gallate (PG) and 9-aminoacridine (9-AA) as inhibitors of camptothecin (CPT)-induced MDC1 foci formation. We demonstrated that the inhibition of CPT-induced MDC1 foci formation by PG was caused by the direct suppression of histone H2AX phosphorylation at Ser139 (γH2AX), which is required for MDC1 foci formation, by quantifying γH2AX in cells and in vitro 9-AA also directly suppressed H2AX Ser139-phosphorylation in vitro but the concentration was much higher than that required to suppress CPT-induced MDC1 foci formation in cells. Consistent with these findings, PG and 9-AA both suppressed CPT-induced G2/M cell-cycle arrest and increased the number of abnormal nuclei. Our results suggest that early DDR-inhibitory effects of PG and 9-AA contribute to their chromosome-damaging potential, and that the EGFP-MDC1 foci formation-inhibition assay is useful for detection of and screening for H2AX Ser139-phosphorylation-inhibitory effects of chemicals.

Individual and combined in vitro (anti)androgenic effects of certain food additives and cosmetic preservatives.

The individual and combined (binary mixtures) (anti)androgenic effect of butylparaben (BuPB), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) was evaluated using the MDA-kb2 cell line. Exposing these cells to AR agonists results in the expression of the reporter gene (encoding for luciferase) and luminescence can be measured in order to monitor the activity of the reporter protein. In case of the evaluation of the anti-androgenic effect, the individual test compounds or binary mixtures were tested in the presence of a fixed concentration of a strong AR agonist (1000 pM 5-alpha-dihydrotestosterone; DHT). Cell viability was assessed using a resazurin based assay. For PG, this is the first report in the literature concerning its (anti)androgenic activity. In case of both individual and mixture testing none of the compounds or binary combinations showed androgenic activity. When tested in the presence of DHT, BuPB, BHA and BHT proved to be weak anti-androgens and this was confirmed during the evaluation of binary mixtures (BuPB+BHA, BuPB+BHT and BHA+BHT). Besides performing the in vitro testing of the binary combinations, two mathematical models (dose addition and response addition) were evaluated in terms of accuracy of prediction of the anti-androgenic effect of the selected binary mixtures. The dose addition model guaranteed a good correlation between the experimental and predicted data. However, no estimation was possible in case of mixtures containing PG, due to the lack of effect of the compound in case of the individual testing.

Geno- and cytotoxicity of propyl gallate food additive.

Synthetic phenolic food additives, such as propyl 3,4,5-trihydroxybenzoate (propyl galate; PG), have been used as an antioxidant in the food industry to prevent oils from spoiling. Their toxicity is one of the challengeable issues resulting from the widespread usage of them in food-related industrials. In this study, we investigated the anticell proliferation effects of PG on A549 lung cancer cells. The result showed that PG dose and time dependently decreased the growth of A549 cells with an half-maximal inhibitory concentration of approximately 1 × 10(-3) and 5 × 10(-4)M of PG at 48 and 72 hours, respectively. In addition, DNA strand breaks have been observed through the comet assay technique. Also, morphology of 4',6-diamidino-2-phenylindole (DAPI)-stained cells showed an obvious fragmentation in the chromatin and DNA rings within the nucleus of PG-treated cells, and, finally, flow cytometry analyses of the cells confirmed DAPI staining assay and determined early and late apoptosis in treated cells.

The relationship between CD86 and CD54 protein expression and cytotoxicity following stimulation with contact allergen in THP-1 cells.

Contact allergens induce the augmentation of cell surface molecules on and release of cytokines from Langerhans cells (LC) in skin sensitization. THP-1 and U937 cell lines, surrogates of LC, were used as analytical tools of this phenomenon recently. In THP-1 cells, contact allergens are reported to induce the phenotypic alteration including the production of pro-inflammatory cytokines and augmentation of cell surface molecules especially at sub-toxic doses. However, the relationship between phenotypic alteration and cytotoxicity is not clear yet. The purpose of this study is to understand the relationship between the protein expression and cytotoxicity induced by contact allergens. First, we observed that the cytotoxicity induced by contact allergens is caused by both apoptosis and necrosis. Apoptosis was preferentially confirmed in stimulation with contact allergens, but non-allergen sodium lauryl sulfate (SLS) hardly induced apoptosis. Moreover, there was no effect to augmentation of protein expression when apoptosis induction pathways were inhibited. Based on these findings, we proposed that the protein expression and cytotoxicity were controlled independently. Next, oxidative stress was found to be generated by contact allergens at the early phase, and this regulated the protein expression and cytotoxicity at least partially. Finally, the humoral factors from dead cells induced by dinitrochlorobenzene (DNCB) were exposed to fresh THP-1 cells to confirm whether protein expression depended on cytotoxicity. The protein expression was not induced. Altogether, these results suggest that cytotoxicity induced by contact allergens may result in apoptosis and may also be stimulated in parallel with protein expression through an intracellular signal or signals.

The enhancement of propyl gallate-induced HeLa cell death by MAPK inhibitors is accompanied by increasing ROS levels.

Propyl gallate (PG) as a synthetic antioxidant exerts a variety of effects on tissue and cell functions. Here, we investigated the effects of MAPK (MEK, JNK and p38) inhibitors on PG-treated HeLa cells in relation to cell death, ROS and GSH levels. PG induced cell growth inhibition and apoptosis in HeLa cells, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm). ROS levels were increased or decreased in PG-treated HeLa cells depending on the incubation times. PG also increased GSH depleted cell numbers in HeLa cells. All the MAPK inhibitors slightly enhanced cell growth inhibition, death and MMP (ΔΨm) loss, and increased ROS levels in PG-treated HeLa cells. However, MAPK inhibitors did not significantly affect GSH depletion in PG-treated cells. In conclusion, the enhanced effect of MAPK inhibitors on PG-induced HeLa cell death was accompanied by increasing ROS levels but the effect was not related to changes of GSH level.

Propyl gallate inhibits the growth of calf pulmonary arterial endothelial cells via glutathione depletion.

Propyl gallate (PG) as a synthetic antioxidant exerts a variety of effects on tissue and cell functions. Here, we evaluated the effects of PG on the growth and death of endothelial cells (ECs), especially calf pulmonary artery endothelial cells (CPAEC) in relation to reactive oxygen species (ROS) and glutathione (GSH). PG dose-dependently inhibited the growth of CPAEC and human umbilical vein endothelial cells (HUVEC) at 24h. PG induced cell death in CPAEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsi(m)). PG generally increased ROS level in CPAEC but not in HUVEC. PG also dose-dependently increased GSH depleted cells in both ECs. The treatment with antioxidant of N-acetyl-cysteine (NAC) or ascorbate acid (AA) prevented CPAEC growth inhibition and death by PG, which was accompanied by the attenuation of GSH depletion but not by the reduction of ROS level. In conclusion, PG induced growth inhibition and death of ECs, especially CPAEC via GSH depletion.

Transcriptome analysis provides new insights into liver changes induced in the rat upon dietary administration of the food additives butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole.

Transcriptomics was performed to gain insight into mechanisms of food additives butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG), and thiabendazole (TB), additives for which interactions in the liver can not be excluded. Additives were administered in diets for 28 days to Sprague-Dawley rats and cDNA microarray experiments were performed on hepatic RNA. BHT induced changes in the expression of 10 genes, including phase I (CYP2B1/2; CYP3A9; CYP2C6) and phase II metabolism (GST mu2). The CYP2B1/2 and GST expression findings were confirmed by real time RT-PCR, western blotting, and increased GST activity towards DCNB. CC altered the expression of 12 genes. Three out of these were related to peroxisomes (phytanoyl-CoA dioxygenase, enoyl-CoA hydratase; CYP4A3). Increased cyanide insensitive palmitoyl-CoA oxidation was observed, suggesting that CC is a weak peroxisome proliferator. TB changed the expression of 12 genes, including CYP1A2. In line, CYP1A2 protein expression was increased. The expression level of five genes, associated with p53 was found to change upon TB treatment, including p53 itself, GADD45alpha, DN-7, protein kinase C beta and serum albumin. These array experiments led to the novel finding that TB is capable of inducing p53 at the protein level, at least at the highest dose levels employed above the current NOAEL. The expression of eight genes changed upon PG administration. This study shows the value of gene expression profiling in food toxicology in terms of generating novel hypotheses on the mechanisms of action of food additives in relation to pathology.

Ecotoxicological effects of the antioxidant additive propyl gallate in five aquatic systems.

Propyl gallate is an antioxidant widely used in foods, cosmetics and pharmaceuticals. The occurrence and fate of additives in the aquatic environment is an emerging issue in environmental chemistry. To date, there is little available information about the adverse effects of propyl gallate on aquatic organisms. Therefore, the toxic effects were investigated, using five model systems from four trophic levels. The most sensitive system was the hepatoma fish cell line PLHC-1 according to total protein content, with an EC(50) of 10 microM and a NOAEL of 1 microM at 72 h, followed by the immobilization of Daphnia magna, the inhibition of bioluminescence of Vibrio fischeri, the salmonid fish cell line RTG-2 and the inhibition of the growth of Chlorella vulgaris. Although protein content, neutral red uptake, methylthiazol metabolization and acetylcholinesterase activity were reduced in PLHC-1 cells, stimulations were observed for lysosomal function, succinate dehydrogenase, glucose-6-phosphate dehydrogenase and ethoxyresorufin-O-deethylase activities. No changes were observed in metallothionein levels. The main morphological observations were the loss of cells and the induction of cell death mainly by necrosis but also by apoptosis. The protective and toxic effects of propyl gallate were evaluated. General antioxidants and calcium chelators did not modify the toxicity of propyl gallate, but an iron-dependent lipid peroxidation inhibitor gave 22% protection. The results also suggest that propyl gallate cytotoxicity is dependent on glutathione levels, which were modulated by malic acid diethyl ester and 2-oxothiazolidine-4-carboxylic acid. According to the results, propyl gallate should be classified as toxic to aquatic organisms.

Final report on the amended safety assessment of Propyl Gallate.

Propyl Gallate is the n-propyl ester of gallic acid (3,4,5-trihydroxybenzoic acid). It is soluble in ethanol, ethyl ether, oil, lard, and aqueous solutions of polyethylene glycol (PEG) ethers of cetyl alcohol, but only slightly soluble in water. Propyl Gallate currently is used as an antioxidant in a reported 167 cosmetic products at maximum concentrations of 0.1%. Propyl Gallate is a generally recognized as safe (GRAS) antioxidant to protect fats, oils, and fat-containing food from rancidity that results from the formation of peroxides. Data on dermal absorption are not available, but Propyl Gallate is absorbed when ingested, then methylated, conjugated, and excreted in the urine. The biological activity of Propyl Gallate is consistent with its free-radical scavenging ability, with effects that include antimicrobial activity, enzyme inhibition, inhibition of biosynthetic processes, inhibition of the formation of nitrosamines, anesthesia, inhibition of neuromuscular response to chemicals, ionizing/ultraviolet (UV) radiation protection, chemoprotection, antimutagenesis, anticarcinogenesis and antitumorigenesis, antiteratogenesis, and anticariogenesis. Animal toxicity studies indicate that Propyl Gallate was slightly toxic when ingested, but no systemic effects were noted with dermal application. Propyl Gallate is a strong sensitizer when tested intradermally, less sensitizing when tested topically, and nonsensitizing topically at 0.1% in one study. In a second study, Propyl Gallate (15 mg dissolved in 8 ml vehicle) was sensitizing to guinea pigs. Acute eye irritation tests conducted on nine cosmetic formulations, each containing less than 1% Propyl Gallate, were negative. A phototoxicity study conducted on a cosmetic formulation containing 0.003% Propyl Gallate determined that the product was not phototoxic to guinea pigs. In one study, female rats fed 0.5 g Propyl Gallate had substantially increased fetal resorption rates when compared to controls, but in four other studies, Propyl Gallate at doses up to 2.04 g/kg was nonteratogenic in rats, rabbits, mice, and hamsters. In clinical cumulative irritancy tests, Propyl Gallate was nonirritating at concentrations up to 10%. Patch tests at concentrations less than 1% yielded positive elicitation responses. Repeat-insult patch tests using cosmetic formulations with 0.003% Propyl Gallate produced no irritation or sensitization. Propyl Gallate at a concentration of 10% in alcohol was nonphototoxic in 25 subjects. Cosmetic formulations, each containing 0.003% Propyl Gallate, produced no signs of photosensitization or phototoxicity in a total of 371 subjects. Although Propyl Gallate is not a skin irritant in clinical tests, the available data demonstrate that it is a skin sensitizer and that it may be a sensitizer at lower concentrations than originally thought, i.e., at concentrations less than 1%. In actual practice, cosmetic formulations contain Propyl Gallate at concentrations up to 0.1% and usage has increased over the past 20 years. In spite of the increased exposure associated with increased use, it is the clinical experience of the Panel that the use of Propyl Gallate in cosmetics has not resulted in sensitization reactions. Therefore, the Panel believes that a concentration limitation of 0.1% in cosmetics is necessary (given the evidence of sensitization at concentrations less than 1%) and sufficient (given that current products are not producing adverse reactions).

Promoting effects of combined antioxidant and sodium nitrite treatment on forestomach carcinogenesis in rats after initiation with N-methyl-N'-nitro-N-nitrosoguanidine.

The effects of sodium nitrite (NaNO2), in combination with one of three antioxidants, tert-butylhydroquinone (TBHQ), alpha-tocopherol (alpha-Toc) and propyl gallate (PG), on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) stomach carcinogenesis were investigated in F344 rats. Groups of 15 male rats were treated with an intragastric dose of 150 mg/kg body weight of MNNG, and starting 1 week later, were treated with 0.5% TBHQ, 1% alpha-Toc, 1% PG or basal diet with or without 0.2% NaNO2 in their drinking water until they were killed at the end of week 36. Macroscopically, in MNNG-treated animals, combined administration of alpha-Toc or PG with NaNO2 significantly increased the areas and numbers of forestomach nodules as compared with the respective antioxidant alone values. Microscopically, in MNNG-treated animals, treatment with TBHQ significantly increased the incidence and multiplicity of forestomach papillomas as compared with basal diet alone value. Combined administration of alpha-Toc with NaNO2 significantly raised the multiplicity of forestomach papillomas, with a tendency to elevation in the incidence as compared with the group given alpha-Toc alone. Incidences of forestomach moderate and/or severe hyperplasias were significantly higher in the TBHQ or PG plus NaNO2 groups than in the single compound groups. In rats without MNNG treatment, combined treatment of antioxidants with NaNO2 significantly increased the incidences of mild or moderate hyperplasia. In the glandular stomach, although the incidence of atypical hyperplasia showed a non-significant tendency for decrease with TBHQ treatment, additional administration of NaNO2 caused significant increase. These results indicate that co-administration of NaNO2 with alpha-Toc, TBHQ or PG and particularly the first, promotes forestomach carcinogenesis. Concurrent alpha-Toc, TBHQ or PG treatment with NaNO2 is likely to induce forestomach tumors in the long term.

Cytogenetic effects of propyl gallate in CHO-K1 cells.

We investigated whether propyl gallate (PG) can induce sister-chromatid exchanges (SCEs) and chromosomal aberrations (CAs) in CHO-K1 cells. In the absence of an exogeneous metabolizing system, treatments with 0.25-1.5mM PG in plugged flasks for 3h resulted in increases in SCEs, CAs, and endoreduplications (ERDs), which were followed by an increase in the percentage of cells showing cell-cycle delay. At the end of the treatment, a decrease in PG concentration and production of PG dimer and ellagic acid (EA) in the medium were detected, indicating that PG had autoxidized. EA, an oxide of PG, was not genotoxic even at 0.3mM, the maximum concentration soluble in the medium. Several oxygen radical scavengers (superoxide dismutase (SOD), catalase, glutathione and o-phenanthroline (OP)) and an inhibitor of catalase activity (3-amino-1,2,4-triazole (AT)), did not significantly influence PG genotoxicity. When PG autoxidation was suppressed by low pH (6.8) or a 5% CO(2) atmosphere, cell-cycle delay intensified and induction of SCEs and CAs occurred even at the lowest PG dose (0.1mM). When PG (0.5mM) was assayed in the presence of S9 (1.5-9%), gallic acid (GA), a metabolite of PG, was generated in direct proportion to the S9 concentration, while cell-cycle delay and genotoxic effects varied inversely with S9 concentration at the levels over 3%. GA also autoxidized and at >or=0.5mM it induced SCEs. Both catalase and AT suppressed the induction of SCEs by GA or inhibited cell proliferation, indicating that H(2)O(2) participated in the effects. In conclusion, PG in the presence or absence of S9 can induce SCEs, CAs, and ERDs, and the oxides, metabolites and oxygen-free radicals generated during the treatment are partly responsible for these effects.

Induction of lipid peroxidation in human fibroblasts by the antioxidant propyl gallate in combination with copper(II).

The antioxidant propyl gallate (PG) induced lipid peroxidation in combination with non-toxic Cu(II) concentrations in human fibroblasts. This was measured by the thiobarbituric acid assay (TBA assay) and by detection of accumulating fluorescent products after a 1-h treatment of cells with CuCl2/PG at concentrations higher than 0.125 mM. PG alone led to a significant reduction of thiobarbituric acid-reactive substances (TBARS) demonstrating its antioxidative properties. Time course studies of lipid peroxidation by PG/Cu(II) showed that formation of TBARS was preceded by a lag phase of 60 min. Thereafter, the TBARS value increased rapidly for 1 h and then reached a constant maximum or slightly decreased. The induction of lipid peroxidation by PG/Cu(II) is probably due to the formation of reactive species like reactive oxygen species (ROS), Cu(I) and semiquinone radicals which are able to participate in initiation and propagation of lipid peroxidation. Combination effects of PG/Cu(II) were demonstrated also on inhibition of membrane-bound succinate dehydrogenase. Cytosolic esterases were affected only slightly. The greater susceptibility of membrane-bound enzymes is in accordance with the lipid peroxidation-inducing effects of PG/Cu(II).

DNA strand break induction and enhanced cytotoxicity of propyl gallate in the presence of copper(II).

The antioxidant propyl gallate (PG) induced single strand breaks in PM2 DNA at concentrations higher than 0.25 microM when it was combined with copper concentrations at 5 microM and above. In combination with 100 microM CuCl2, extensive double strand breakage was also observed. Neither PG alone nor CuCl2 showed any strand breaking properties. DNA strand breakage was inhibited by addition of catalase or the Cu(I) chelator neocuproine, indicating the involvement of H2O2 and a Cu(II)/Cu(I) redox cycle in the DNA damage. DNA damage of PG/Cu(II) was also observed in human fibroblasts. Using the alkaline elution technique concentrations of 0.15-0.5 mM PG induced DNA strand breaks in combination with 2.5 mM CuCl2, while the single substances did not show any effect. At these concentrations cell viability measured by the MTT assay was not reduced by more than 10%; however, cell growth was inhibited by PG in combination with Cu(II). This growth inhibition was apparently due to the DNA damage incurred by PG/Cu(II). The synergistic interaction between PG and Cu(II) is probably caused by a redox reaction between both compounds, whereby reactive species such as ROS are formed, which are responsible for the observed genotoxic and cytotoxic effects. Our results demonstrate that the antioxidative and cytoprotective properties of propyl gallate may change to prooxidative, cytotoxic and genotoxic properties in the presence of Cu(II).