Reproductive and endocrine-disrupting toxicity of pyrogallol in catfish (Clariasgariepinus).

Endocrine disruptors are synthetic or natural chemicals that can agonize/antagonize hormone receptors or can interfere with the production and secretion of hormones, leading to altered tissue histology and physiology. Pyrogallol is a contaminant widely distributed in aquatic environments that presents health risks to both humans and animals. However, the potential for endocrine disruption by pyrogallol, particularly in fish, are lacking. The purpose of this study was to shed light on how pyrogallol may affect hormone signalling, histopathology, and reproductive outcomes in African catfish Clarias gariepinus. To investigate this, African catfish were exposed to one sublethal concentration of pyrogallol at either 0, 1, 5 or 10 mg/L for 15 days. We then assessed the effects of pyrogallol on the thyroid gland as well as the reproductive system by measuring sex hormone, seminal quality, gonadal histopathology, and histochemistry. Thyroid stimulating hormone and thyroxine showed notable decreases in catfish, and triiodothyronine was decreased with 10 mg/L pyrogallol. Unlike luteinizing hormone, follicle-stimulating hormone was significantly reduced in fish following exposure to pyrogallol relative to controls. Testosterone was also decreased in fish following pyrogallol exposure, whereas 17ß-estradiol increased in catfish exposed to pyrogallol. Additionally, in response to pyrogallol toxicity, sperm quality indices, including count, spermatocrit, motility, and sperm viability were adversely affected in a concentration-dependent manner. Pyrogallol exposure also induced several changes in the gonad following exposure to 1, 5, or 10 mg/L. Deformed tubular structures, vacuolation, thickening of the basement membrane, hypertrophy of the seminiferous tubules, intense melanomacrophage localization, spermatozoa loss, and necrosis were all observed in the testes. In the ovary, atretic follicles, deteriorated mature oocytes, degenerated yolk globules, and an increase in perinucleolar oocytes were observed in catfish exposed to pyrogallol. These findings suggest that pyrogallol may act as endocrine disrupting substance in aquatic environments. Further research on the mechanisms by which pyrogallol impairs endocrine systems, particularly in fish, is recommended.

Immunotoxicological, histopathological, and ultrastructural effects of waterborne pyrogallol exposure on African catfish (Clariasgariepinus).

Pyrogallol is a naturally occurring polyphenol derived from natural plants, such as Acer rubrum and Eucalyptus sp. The current study was designed to evaluated pyrogallol-mediated toxicity at sublethal levels (1, 5, and 10 mg/L), derived from 96 h-LC50 values previously determined for African catfish (Clarias gariepinus). Immunotoxicological indices, histological, histochemical, and ultrastructural alterations in C. gariepinus were evaluated following a 15-day pyrogallol exposure. Pyrogallol decreased immune parameters [lysozyme activity (LYZ), immunoglobulin M (IgM), and phagocytic activity] and increased pro-inflammatory cytokines, interleukin-1 beta (IL-1ß), interleukin-6 (IL-6) in the serum of C. gariepinus. In addition, histopathology analysis demonstrated that exposure to pyrogallol induced injury in the liver and spleen of fish. Cellular changes in the liver include hepatocyte hydropic degeneration, melanomacrophage, vacuolated hepatocytes, congested blood, severe structural deformation, and hemorrhage. In the spleen, ellipsoid structures, melanomacrophage centers, and infiltration of inflammatory cells were evident. Together, a high frequency of histopathological lesions was scored in both the liver and spleen of C. gariepinus, which showed a dose-dependent relationship between pyrogallol exposure and histopathological indices. Our data suggest that dysfunction in the immune system may be mediated by pyrogallol-induced changes in cytokines.

Exposure to pyrogallol impacts the hemato-biochemical endpoints in catfish (Clarias gariepinus).

Pyrogallol is widely used in several industrial applications and can subsequently contaminate aquatic ecosystems. Here, we report for the first time the presence of pyrogallol in wastewater in Egypt. Currently, there is a complete lack of toxicity and carcinogenicity data for pyrogallol exposure in fish. To address this gap, both acute and sub-acute toxicity experiments were conducted to determine the toxicity of pyrogallol in catfish (Clarias gariepinus). Behavioral and morphological endpoints were evaluated, in addition to blood hematological endpoints, biochemical indices, electrolyte balance, and the erythron profile (poikilocytosis and nuclear abnormalities). In the acute toxicity assay, it was determined that the 96 h median-lethal concentration (96 h-LC50) of pyrogallol for catfish was 40 mg/L. In sub-acute toxicity experiment, fish divided into four groups; Group 1 was the control group. Group 2 was exposed to 1 mg/L of pyrogallol, Group 3 was exposed to 5 mg/L of pyrogallol, and Group 4 was exposed to 10 mg/L of pyrogallol. Fish showed morphological changes such as erosion of the dorsal and caudal fins, skin ulcers, and discoloration following exposure to pyrogallol for 96 h. Exposure to 1, 5, or 10 mg/L pyrogallol caused a significant decrease in hematological indices, including red blood cells (RBCs), hemoglobin, hematocrit, white blood cells (WBC), thrombocytes, and large and small lymphocytes in a dose-dependent manner. Several biochemical parameters (creatinine, uric acid, liver enzymes, lactate dehydrogenase, and glucose) were altered in a concentration dependent manner with short term exposures to pyrogallol. Pyrogallol exposure also caused a significant concentration-dependent rise in the percentage of poikilocytosis and nuclear abnormalities of RBCs in catfish. In conclusion, our data suggest that pyrogallol should be considered further in environmental risk assessments of aquatic species.

Chemical Composition and Preliminary Toxicity Evaluation of the Essential Oil from Peperomia circinnata Link var. circinnata. (Piperaceae) in Artemia salina Leach.

Peperomia Ruiz and Pav, the second largest genus of the Piperaceae, has over the years shown potential biological activities. In this sense, the present work aimed to carry out a seasonal and circadian study on the chemical composition of Peperomia circinata essential oils and aromas, as well as to evaluate the preliminary toxicity in Artemia salina Leach and carry out an in silico study on the interaction mechanism. The chemical composition was characterized by gas chromatography (GC/MS and GC-FID). In the seasonal study the essential oil yields had a variation of 1.2-7.9%, and in the circadian study the variation was 1.5-5.6%. The major compounds in the seasonal study were ß-phellandrene and elemicin, in the circadian they were ß-phellandrene and myrcene, and the aroma was characterized by the presence of ß-phellandrene. The multivariate analysis showed that the period and time of collection influenced the essential oil and aroma chemical composition. The highest toxicity value was observed for the essential oil obtained from the dry material, collected in July with a value of 14.45 ± 0.25 µg·mL-1, the in silico study showed that the major compounds may be related to potential biological activity demonstrated by the present study.

Role of Metabolic Activation in Elemicin-Induced Cellular Toxicity.

Elemicin, an alkenylbenzene constituent of natural oils of several plant species, is widely distributed in food, dietary supplements, and medicinal plants. 1'-Hydroxylation is known to cause metabolic activation of alkenylbenzenes leading to their potential toxicity. The aim of this study was to explore the relationship between elemicin metabolism and its toxicity through comparing the metabolic maps between elemicin and 1'-hydroxyelemicin. Elemicin was transformed into a reactive metabolite of 1'-hydroxyelemicin, which was subsequently conjugated with cysteine (Cys) and N-acetylcysteine (NAC). Administration of NAC could significantly ameliorate the elemicin- and 1'-hydroxyelemicin-induced cytotoxicity of HepG2 cells, while depletion of Cys with diethyl maleate (DEM) increased cytotoxicity. Recombinant human CYP screening and CYP inhibition experiments revealed that multiple CYPs, notably CYP1A1, CYP1A2, and CYP3A4, were responsible for the metabolic activation of elemicin. This study revealed that metabolic activation plays a critical role in elemicin cytotoxicity.

Metabolic Activation of Myristicin and Its Role in Cellular Toxicity.

Myristicin is widely distributed in spices and medicinal plants. The aim of this study was to explore the role of metabolic activation of myristicin in its potential toxicity through a metabolomic approach. The myristicin- N-acetylcysteine adduct was identified by comparing the metabolic maps of myristicin and 1'-hydroxymyristicin. The supplement of N-acetylcysteine could protect against the cytotoxicity of myristicin and 1'-hydroxymyristicin in primary mouse hepatocytes. When the depletion of intracellular N-acetylcysteine was pretreated with diethyl maleate in hepatocytes, the cytotoxicity induced by myristicin and 1'-hydroxymyristicin was deteriorated. It suggested that the N-acetylcysteine adduct resulting from myristicin bioactivation was closely associated with myristicin toxicity. Screening of human recombinant cytochrome P450s (CYPs) and treatment with CYP inhibitors revealed that CYP1A1 was mainly involved in the formation of 1'-hydroxymyristicin. Collectively, this study provided a global view of myristicin metabolism and identified the N-acetylcysteine adduct resulting from myristicin bioactivation, which could be used for understanding the mechanism of myristicin toxicity.

Microbial metabolites of proanthocyanidins reduce chemical carcinogen-induced DNA damage in human lung epithelial and fetal hepatic cells in vitro.

Seven selected microbial metabolites of proanthocyanidins (MMP), 3-phenylpropionic, 4-hydroxyphenyl acetic, 3-(4-hydroxyphenyl) propionic, p-coumaric, benzoic acid, pyrogallol (PG), and pyrocatechol (PC) were evaluated for their ability to reduce chemical carcinogen-induced toxicity in human lung epithelial cells (BEAS-2B) and human fetal hepatic cells (WRL-68). Cells pre-treated with MMP were exposed to a known chemical carcinogen, 4-[(acetoxymethyl) nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) to assess MMP-mediated cytoprotection and reduction of DNA damage. PG in BEAS-2B and PC in WRL-68 cells mitigated the NNKOAc-induced cytotoxicity. Pre-incubation of PG depicted significant protection against NNKOAc-induced DNA damage in BEAS-2B cells. PC in WRL-68 cells showed similar activity. To understand the mechanisms of PG- and PC-mediated DNA damage reduction, the effect on DNA damage response (DDR) proteins, cellular reactive oxygen species (ROS), total antioxidant capacity (TAC), glutathione peroxidase (GPx), and caspase activity were studied. PG and PC alter the DDR and may promote ATR-Chk1 and ATM-Chk2 pathways, respectively. Cellular oxidative stress induced by NNKOAc was mitigated by PG and PC through enhanced GPx expression and TAC. PG and PC suppressed the activation of the extrinsic apoptotic pathway (caspase 3 and 8) provoked by NNKOAc. MMP are beneficial in chemoprevention by reducing cellular DNA damage.

Insecticidal Activity of the Leaf Essential Oil of Peperomia borbonensis Miq. (Piperaceae) and Its Major Components against the Melon Fly Bactrocera cucurbitae (Diptera: Tephritidae).

The essential oil from the leaves of Peperomia borbonensis from Réunion Island was obtained by hydrodistillation and characterized using GC-FID, GC/MS and NMR. The main components were myristicin (39.5%) and elemicin (26.6%). The essential oil (EO) of Peperomia borbonensis and its major compounds (myristicin and elemicin), pure or in a mixture, were evaluated for their insecticidal activity against Bactrocera cucurbitae (Diptera: Tephritidae) using a filter paper impregnated bioassay. The concentrations necessary to kill 50% (LC50 ) and 90% (LC90 ) of the flies in three hours were determined. The LC50 value was 0.23 ± 0.009 mg/cm2 and the LC90 value was 0.34 ± 0.015 mg/cm2 for the EO. The median lethal time (LT50 ) was determined to compare the toxicity of EO and the major constituents. The EO was the most potent insecticide (LT50  = 98 ± 2 min), followed by the mixture of myristicin and elemicin (1.4:1) (LT50  = 127 ± 2 min) indicating that the efficiency of the EO is potentiated by minor compounds and emphasizing one of the major assets of EOs against pure molecules.

Effects of pyrogallic acid on Microcystis aeruginosa: oxidative stress related toxicity.

Pyrogallic acid (PA) is used in various industrial and consumer products. The molecular mechanisms underlying PA's toxicity was not fully understood. In this study, toxicity of PA on Microcystis aeruginosa with reactive oxygen species (ROS) generation as an end point was investigated. The results showed an increase in the percentage of cells with loss of membrane integrity and enhanced intracellular ROS production. Exposure to 50mgL(-1) PA for 48h caused the highest percentage of loss of membrane integrity (56.7%), and a 2.54-fold higher intracellular ROS level compared to control. Further investigation revealed that PA caused a dose-dependent increase in DNA strand breaks (DSB) of M. aeruginosa at exposure concentration from 2 to 50mgL(-1). The incubation of cells with ROS scavengers ascorbic acid, N-acetyl-l-cysteine (NAC) and tocopherol markedly alleviated the level of PA-induced DSB. Analysis of PA autoxidized products in culture solution showed that PA was quickly converted to purpurogallin (PG), and PG was further autoxidized to other polyphenolic compounds. PA and PG might participate a futile redox cycle, which mediated ROS production in M. aeruginosa. These results suggested DNA strands and cell membrane were two targets of ROS induced by PA, and oxidative damage was an important mechanism for the toxicity of PA against M. aeruginosa.

Toxicology and carcinogenesis studies of pyrogallol (CAS No. 87-66-1) in F344/N rats and B6C3F1/N mice (dermal studies).

UNLABELLED: The current main commercial use of pyrogallol is the production of pharmaceuticals and pesticides. In analytical chemistry, pyrogallol is used as a complexing agent, reducing agent, and, in alkaline solution, as an indicator of gaseous oxygen. Pyrogallol was nominated for testing by private individuals based on its frequent occurrence in natural and manufactured products, including hair dyes, and the apparent lack of carcinogenicity data. Male and female F344/N rats and B6C3F1/N mice were administered pyrogallol (99% pure) dermally for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, mouse bone marrow cells, and mouse peripheral blood erythrocytes. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats received dermal applications of pyrogallol in 95% ethanol at doses of 0, 9.5, 18.75, 37.5, 75, or 150 mg pyrogallol/kg body weight, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female special study rats were administered the same doses, 5 days per week for 23 days. All rats survived until the end of the study except for one vehicle control female. Mean body weights of dosed groups of males and females were generally similar to those of the vehicle controls. Chemical-related clinical findings included brown staining and irritation of the skin at the site of application. There were no changes in the hematology, serum clinical chemistry, thyroid hormone values, or organ weights attributable to the dermal administration of pyrogallol. The incidences of squamous hyperplasia, hyperkeratosis, and chronic active inflammation of the skin at the site of application were significantly increased in all dosed groups of males and females. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice received dermal applications of pyrogallol in 95% ethanol at doses of 0, 38, 75, 150, 300, or 600 mg pyrogallol/kg body weight, 5 days per week for 14 weeks. All mice survived until the end of the study. Mean body weights of dosed groups of males and females were similar to those of the vehicle controls. Chemical-related clinical findings included brown staining and irritation at the site of application. There were no changes in the hematology values or organ weights attributable to the dermal administration of pyrogallol. The incidences of squamous hyperplasia, hyperkeratosis, and chronic active inflammation of the skin at the site of application were significantly increased in all dosed groups of males and females. The incidence of hematopoietic cell proliferation of the spleen in 600 mg/kg males was significantly greater than that in the vehicle control group. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats received dermal applications of pyrogallol in 95% ethanol at doses of 0, 5, 20, or 75 mg pyrogallol/kg body weight, 5 days per week for up to 104 weeks. Survival of dosed groups of male and female rats was similar to that of the vehicle control groups. Mean body weights of dosed male and female rats were similar to those of the vehicle control groups throughout the study. Irritation of the skin at the site of application was the only chemical-related clinical finding and occurred in the 20 and 75 mg/kg groups. In the skin at the site of application, there were significant increases in the incidences of hyperplasia in all dosed groups of males and females, hyperkeratosis in 20 and 75 mg/kg males and all dosed groups of females, inflammation in 75 mg/kg males and 20 and 75 mg/kg females, and sebaceous gland hyperplasia in 20 and 75 mg/kg males and females. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice received dermal applications of pyrogallol in 95% ethanol at doses of 0, 5, 20, or 75 mg pyrogallol/kg body weight, 5 days per week for up to 105 weeks. Survival of dosed groups of male mice was similar to that of the vehicle control group. Survival was significantly decreased in 75 mg/kg females; most early deaths in this group were due to ulcers at or adjacent to the site of application. The mean body weights of 75 mg/kg female mice were generally over 10% less than those of the vehicle controls during year 2 of the study. Irritation and/or ulceration of the skin at the site of application were the only chemical-related clinical findings and occurred predominantly in the 20 and 75 mg/kg groups. In the skin at the site of application, the incidence of squamous cell carcinoma in 75 mg/kg females was significantly greater than that in the vehicle control group. Two 75 mg/kg males had squamous cell papillomas; squamous cell papillomas have not been observed in historical control male mice in four ethanol dermal studies. Increased incidences of nonneoplastic lesions at the site of application included hyperplasia and hyperkeratosis in all dosed groups; inflammation, fibrosis, and pigmentation in the 20 and 75 mg/kg groups; and sebaceous gland hyperplasia and ulcer in the 75 mg/kg groups. Similar lesions in the skin of the neck and back immediately adjacent to the site of application were observed; the incidences of hyperplasia, hyperkeratosis, ulcer, inflammation, and fibrosis at these sites were significantly increased in 75 mg/kg male and female mice, and the incidence of sebaceous gland hyperplasia was significantly increased in 75 mg/kg female mice. Dermal application of pyrogallol also resulted in significant increases in the incidences of bone marrow hyperplasia in males and females and lymphoid hyperplasia of the axillary, inguinal, and mandibular lymph nodes; adrenal cortical hematopoietic cell proliferation; and mammary gland hyperplasia in females. GENETIC TOXICOLOGY: Pyrogallol was tested in two independent bacterial mutation studies; both studies gave positive results in one or more strains of S. typhimurium or E. coli. In the first study, positive results were seen in S. typhimurium strain TA100 with and without S9 exogenous metabolic activation, and negative results were obtained in strain TA98. In the second study, which was conducted with the same lot of pyrogallol that was used in the 3-month and 2-year studies, positive results were obtained in S. typhimurium strains TA98, TA100, and in E. coli strain WP2 uvrA/pKM101 in the absence of S9. With S9, this sample of pyrogallol was mutagenic in the E. coli strain but gave equivocal responses in S. typhimurium strains TA98 and TA100. In vivo, a micronucleus test that measured frequency of micronucleated polychromatic erythrocytes in bone marrow of male B6C3F1/N mice following three intraperitoneal injections of pyrogallol, gave negative results. In a second in vivo test, no increase in the frequency of micronucleated erythrocytes was observed in the peripheral blood of female B6C3F1/N mice treated with pyrogallol via dermal application for 3 months; in male mice, however, results were equivocal, based on a significant increase in micronucleated erythrocytes observed at a single dose level at the end of the 3-month study. CONCLUSIONS: Under the conditions of these 2-year dermal studies, there was no evidence of carcinogenic activity of pyrogallol in male or female F344/N rats administered 5, 20, or 75 mg/kg. There was equivocal evidence of carcinogenic activity of pyrogallol in male B6C3F1/N mice based on increased incidences of squamous cell papilloma of the skin at the site of application. There was some evidence of carcinogenic activity of pyrogallol in female B6C3F1/N mice based on increased incidences of squamous cell carcinoma of the skin at the site of application. Dermal administration of pyrogallol caused increased incidences of nonneoplastic lesions of the skin at the site of application in male and female rats and mice, skin adjacent to the site of application in male and female mice, and mammary gland in female mice.

Pyrogallol-associated dermal toxicity and carcinogenicity in F344/N rats and B6C3F1/N mice.

Pyrogallol (CAS No. 87-66-1), a benzenetriol used historically as a hair dye and currently in a number of industrial applications, was nominated to the National Toxicology Program (NTP) for testing based on the lack of toxicity and carcinogenicity data. Three-month and two-year toxicity studies to determine the toxicity and carcinogenicity of pyrogallol when applied to naïve skin (i.e. dermal administration) were conducted in both sexes of F344/N rats and B6C3F1/N mice. In the three-month studies, adult rodents were administered pyrogallol in 95% ethanol five days per week for 3 months at doses of up to 150 mg/kg body weight (rats) or 600 mg/kg (mice). Based on the subchronic studies, the doses for the two-year studies in rats and mice were 5, 20 and 75 mg/kg of pyrogallol. All mice and most rats survived until the end of the three-month study and body weights were comparable to controls. During the two-year study, survival of dosed rats and male mice was comparable to controls; however survival of 75 mg/kg female mice significantly decreased compared to controls. The incidences of microscopic non-neoplastic lesions at the site of application were significantly higher in all dosed groups of rats and mice and in both the 3-months and two-year studies. In the two-year study, hyperplasia, hyperkeratosis and inflammation tended to be more severe in mice than in rats, and in the mice they tended to be more severe in females than in males. The incidence of squamous cell carcinoma at the site of application (SOA) in 75 mg/kg female mice and SOA squamous cell papillomas in 75 mg/kg male mice were greater than controls. Pyrogallol was carcinogenic in female mice and may have caused tumors in male mice.

Toxicological actions of plant-derived and anthropogenic methylenedioxyphenyl-substituted chemicals in mammals and insects.

The methylenedioxyphenyl (MDP) substituent is a structural feature present in many plant chemicals that deter foraging by predatory insects and herbivores. With increasing use of herbal extracts in alternative medicine, human exposure to MDP-derived plant chemicals may also be significant. Early studies found that most MDP agents themselves possess relatively low intrinsic toxicity, but strongly influence the actions of other xenobiotics in mammals and insects by modulating cytochrome P-450 (CYP)-dependent biotransformation. Thus, after exposure to MDP chemicals an initial phase of CYP inhibition is followed by a sustained phase of CYP induction. In insects CYP inhibition by MDP agents underlies their use as pesticide synergists, but analogous inhibition of mammalian CYP impairs the clearance of drugs and foreign compounds. Conversely, induction of mammalian CYP by MDP agents increases xenobiotic oxidation capacity. Exposure of insects to MDP-containing synergists in the environment, in the absence of coadministered pesticides, may also enhance xenobiotic detoxication. Finally, although most MDP agents are well tolerated, several, typified by safrole, aristolochic acid, and MDP-kavalactones, are associated with significant toxicities, including the risk of hepatotoxicity or tumorigenesis. Thus, the presence of MDP-substituted chemicals in the environment may produce a range of direct and indirect toxicities in target and nontarget species.

Extract of the plant Cotinus coggygria Scop. attenuates pyrogallol-induced hepatic oxidative stress in Wistar rats.

To examine the protective potential of the Cotinus coggygria Scop. methanol extract, Wistar rats were treated with the hepatotoxic compound pyrogallol, which possesses a potent ability to generate free radicals and induce oxidative stress. The ability of the extract to counteract the oxidative stress was examined in rats that were injected with the extract intraperitoneally (500 mg·(kg body weight)(-1)) either 2 or 12 h before the pyrogallol treatment. The extract possesses a reducing activity in vitro and an ability to chelate the ferrous ion both in vivo and in vitro. Application of the extract prior to pyrogallol treatment led to a decrease in the levels of thiobarbituric acid-reactive substances, aspartate aminotransferase, and alanine aminotransferase, increased activities of antioxidant enzymes and attenuation of DNA damage, as well as increased Akt activity and inhibition of NF-κB protein expression. Treatment with the extract 12 h prior to pyrogallol administration was more effective in suppressing pyrogallol-induced oxidative damage than the 2 h pretreatment. Extract administration promoted an increase in acute phase reactants haptoglobin and α(2)-macroglobulin that was short of a full-fledged acute phase response. Administration of the extract considerably improved the markers of oxidative stress, thus revealing a potential hepatoprotective activity. Our results suggest that Akt activation, NF-κB inhibition, and induction of the acute phase play important roles in mediating hepatic protection by the extract. The greater effectiveness of the 12 h pretreatment with extract points to the important role that preconditioning assumes in improving resistance to subsequent exposure to oxidative stress.

Pyrogallol-induced As4.1 juxtaglomerular cell death is attenuated by MAPK inhibitors via preventing GSH depletion.

Pyrogallol (PG) induces apoptosis in several types of cells mediated by superoxide anion (O(2*-)). Here, we investigated the effects of PG and/or MAPK (MEK, JNK, and p38) inhibitors on the changes in cell growth, cell death, reactive oxygen species (ROS), and GSH levels in As4.1 juxtaglomerular (JG) cells. PG inhibited the growth of As4.1 cells. It also induced apoptosis and the loss of mitochondrial membrane potential (MMP; DeltaPsi(m)) and increased the level of p53 protein. Intracellular O2(*-) level was increased in PG-treated As4.1 cells. PG also increased the number of GSH deleted cells in As4.1 cells. All the MAPK inhibitors significantly attenuated the growth inhibition and death mediated by PG. They decreased the levels of p53 protein and MMP (DeltaPsi(m)) loss in PG-treated As4.1 cells. They also reduced O2(*-) level and GSH-depleted cell number in these cells. In conclusion, MAPK inhibitors attenuated As4.1 cell growth inhibition and death mediated by PG treatment. The changes in O2(*-) and GSH levels by PG and/or MAPK inhibitors appeared to affect the growth and death of As4.1 cells.

Pyrogallol-induced calf pulmonary arterial endothelial cell death via caspase-dependent apoptosis and GSH depletion.

Pyrogallol (PG) as a polyphenol induces apoptosis in cells. Here, we evaluated the effects of PG on the growth and death of endothelial cells (ECs). PG dose-dependently inhibited the growth of calf pulmonary artery endothelial cells (CPAEC) and human umbilical vein endothelial cells (HUVEC). PG also induced apoptosis in both cells accompanied by the loss of mitochondrial membrane potential (DeltaPsi(m)). CPAEC were more sensitive to PG than HUVEC concerning cell growth and death. Caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) did not affect the growth inhibition of CPAEC by PG. However, pan-caspase inhibitor (Z-VAD) significantly reduced apoptosis and the loss of DeltaPsi(m) in PG-treated CPAEC. PG reduced ROS level and increased GSH depleted cell numbers in CPAEC. While Z-VAD increased ROS levels in PG-treated CPAEC, it decreased GSH depleted cell numbers. In conclusion, PG inhibited the growth of ECs, especially CPAEC via caspase-dependent apoptosis and GSH depletion.

Pyrogallol induces G2-M arrest in human lung cancer cells and inhibits tumor growth in an animal model.

Pyrogallol, a catechin compound, is an active component of Emblica officinalis extracts and has an anti-proliferative effect on some human cancer cell lines. In our preliminary study, pyrogallol had highly cytotoxic effect on human lung cancer cell lines and less effect on human bronchial epithelium cell line. This study was performed to investigate the beneficial effect of pyrogallol on human lung cancer cell lines - H441 (lung adenocarcinoma) and H520 (lung squamous cell carcinoma). The MTT (cytotoxic) data showed the inhibition growth of lung cancer cells followed pyrogallol treatment. The cell cycle of lung cancer cells was arrested in G2/M phase using flow cytometry. Using Western blot analysis, the cell cycle related proteins - cyclin B1 and Cdc25c were decreased in a time-dependent manner and the phosphorylated Cdc2 (Thr14) was increased within 4h pyrogallol treatment. Moreover, the higher cleavage of poly (ADP)-ribose polymerase (PARP), the increased of Bax concurrent with the decreased of Bcl-2 indicated that pyrogallol treatment resulted in apoptosis of lung cancer cells. The cell apoptosis was also directly demonstrated using Annexin V-FITC and TUNEL stain. Additionally, the tumoricidal effect of pyrogallol was measured using a xenograft nude mice model. After 5 weeks of pyrogallol treatment could cause the regression of tumor. Taken in vitro and in vivo studies together, these results suggest that pyrogallol can be developed as a promising anti-lung cancer drug particular for the non-small cell lung cancer (NSCLC).

Cytotoxic activity of 3,3',4,4',5,5'-hexahydroxystilbene against breast cancer cells is mediated by induction of p53 and downregulation of mitochondrial superoxide dismutase.

The phytochemical resveratrol, which is found in grapes and red wine, has been reported to have a variety of biological properties. It was shown in our previous research that introduction of additional hydroxyl groups into the stilbene structure increases the biological activity of resveratrol. In this study, the activity of 3,3',4,4',5,5'-hexahydroxystilbene (M8) was investigated in ZR-75-1, MDA-MB-231 and T47D human breast cancer cells. For evaluation of cytotoxic activity of M8, clonogenic and cell proliferation assays were used. The IC50 values obtained in the clonogenic assay were 0.846 microM for T47D, 8.53 microM for ZR-75-1 cells and 25.5 microM for MDA-MB-231, while IC50 values obtained in the cell proliferation assay were significantly higher: 90.1 microM, 98.4 microM, 127.8 microM for T47D, ZR-75-1 and MDA-MB-231 cells, respectively. Compound M8 caused the activation of caspase-8 in MDA-MB-231 cells (marker of extrinsic apoptotic pathway), while activities of caspase-9 (marker of intrinsic apoptotic pathway) and caspase-3 were increased in all 3 tested cell lines. Activation of caspase-9 and caspase-3 was connected with loss of mitochondrial potential and increase of p53, which could have an impact on downregulation of mitochondrial superoxide dismutase (MnSOD) seen in our experiments. MnSOD is a key enzyme providing antioxidative defense in mitochondria - the cellular center of reactive oxygen species' generation. Downregulation of MnSOD can therefore cause a significant decrease of antioxidant defense in cancer cells. An increase of oxidative stress conditions was suggested by loss of reduced glutathione in tested cells. Since cancer cells are usually under permanent oxidative stress, additional increased ROS generation as a result of the interaction of M8 with the mitochondrial respiratory chain and a decrease in oxidative defense can therefore be a promising method for selective elimination of cancer cells.

Apoptosis in pyrogallol-treated Calu-6 cells is correlated with the changes of intracellular GSH levels rather than ROS levels.

We investigated the involvement of glutathione (GSH) and reactive oxygen species (ROS) such as H2O2 and O2-* in the deaths of pyrogallol-treated Calu-6 cells. Pyrogallol inhibited the growth of Calu-6 cells with an IC50 of approximately 50 microM. Levels of intracellular H2O2 were not altered or were decreased in pyrogallol-treated Calu-6 cells at 72 h. However, levels of O2*- were increased. Treatment with pyrogallol also reduced the intracellular GSH content. The activity of SOD was down-regulated, but the activity of catalase was up-regulated by pyrogallol at 72 h. ROS scavengers, including Tempol, Tiron, Trimetazidine, and N-acetylcysteine (NAC), did not reduce the levels of the intracellular O2*-. Tempol showing the recovery of GSH depletion in pyrogallol-treated cells significantly prevented apoptosis, while Tiron prevented the loss of mitochondrial transmembrane potential (DeltaPsi(m)). In contrast, treatment with NAC showing an increased effect on O2*- levels and depletion of GSH intensified pyrogallol-induced apoptosis. In addition, treatment with SOD and catalase significantly prevented the loss of mitochondrial transmembrane potential (DeltaPsi(m)) in pyrogallol-treated Calu-6 cells. However, only catalase showing a decreased effect on O2*- levels and depletion of GSH prevented pyrogallol-induced apoptosis. Taken together, apoptosis in pyrogallol-treated Calu-6 cells is correlated with the changes of intracellular GSH levels rather than ROS levels.

Effect of silymarin on pyrogallol- and rifampicin-induced hepatotoxicity in mouse.

Rifampicin and pyrogallol, besides beneficial effects, elicit hepatotoxicity in experimental animals and humans. The present investigation was undertaken to elucidate the role of drug/toxicant-metabolizing enzymes in rifampicin- and pyrogallol-induced hepatotoxicity and the effect of silymarin, a herbal antioxidant, on rifampicin- and pyrogallol-induced alterations in mouse liver. Male Swiss albino mice were treated intraperitoneally with and without rifampicin (20 mg/kg) and/or pyrogallol (40 mg/kg) for 1, 2, 3 and 4 weeks. In some experiments, animals were treated with silymarin (40 mg/kg), 2 h prior to rifampicin and/or pyrogallol. The differential expression and catalytic activity of cytochrome P-450 (CYP) 1A1, CYP1A2 and CYP2E1, the activity of glutathione-S-transferase, glutathione peroxidase and glutathione reductase, and lipid peroxidation were measured in the liver of control and treated groups. CYP1A1 expression and catalytic activity were not altered following individual or combinational treatment. A significant augmentation in the expression and activity of CYP1A2 and CYP2E1 was observed following pyrogallol and rifampicin+pyrogallol treatment; however, rifampicin exhibited a significant induction of CYP2E1 only. Attenuation of glutathione-S-transferase, glutathione reductase and glutathione peroxidase activities and augmentation of lipid peroxidation were observed following rifampicin and/or pyrogallol treatment and a cumulative effect was seen when the two drugs were administered in combination. Silymarin restored the rifampicin- and/or pyrogallol-induced alterations in the expression and activity of CYP1A2 and CYP2E1, the activity of glutathione-S-transferase, glutathione reductase, and glutathione peroxidase, and lipid peroxidation. The results demonstrate the role of CYP1A2, CYP2E1, glutathione-S-transferase, glutathione reductase and glutathione peroxidase in rifampicin- and pyrogallol-induced hepatotoxicity and provide evidence for the involvement of silymarin in attenuation of drug-induced hepatotoxicity.

Pyrogallol, ROS generator inhibits As4.1 juxtaglomerular cells via cell cycle arrest of G2 phase and apoptosis.

Pyrogallol as a catechin compound has been employed as an O(2)(*-) generator and often used to investigate the role of ROS in the biological system. Here, we investigated the in vitro effect of pyrogallol on cell growth, cell cycle and apoptosis in As4.1 juxtaglomerular cells. Dose-dependent inhibition of cell growth was observed with IC(50) of about 60 microM for 48 h using MTT assay. Pyrogallol (100 microM) did not alter intracellular H(2)O(2) level and catalase activity, but increased the intracellular O(2)(-) level and decreased SOD activity in As4.1 cells. DNA flow cytometric analysis indicated that 50 and 100 microM pyrogallol significantly increased G2 phase cells as compared with those of pyrogallol-untreated cells. Also, pyrogallol induced apoptosis as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay and DAPI staining. This apoptosis process was accompanied with the loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bcl-2 decrease, caspase-3 activation and PARP cleavage. Pan caspase inhibitor (Z-VAD) could significantly rescue As4.1 cells from pyrogallol-induced cell death. But, the inhibitors of caspase-3, caspase-8, and caspase-9 did not prevent apoptotic events in pyrogallol-treated As4.1 cells. Taken together, we have demonstrated that an ROS inducer, pyrogallol inhibits the growth of As4.1 JG cells via cell cycle arrest and apoptosis, and suggest that the compound exhibits an anti-proliferative efficacy on these cells.