Inhibition of benzo(a)pyrene-induced carcinogenesis by vitamin C alone and by vitamin C/vitamin E and selenium/glutathione

The reduction on peroxidation caused by benzopyrenes by some naturally occurring antineoplastic agents was studied in this experimental work. Inhibition/reduction of experimental carcinogenesis induced by benzo(a)pyrene by vitamin C alone and by vitamin C/vitamin E and selenium/glutathione was attempted in 224 female Wistar rats divided in four groups. Injected with 10.08 mL benzo(a)pyrene, the animals were treated with some naturally occurring substances like vitamin C alone and a combination of anticarcinogens. By calculating the carcinogenic potency of benzo(a)pyrene and the anticarcinogenic potency of substances used as well as histological examination of developed tumors and survival time of treated animals, it was found that vitamin C exerts a significant anticarcinogenic effect of 8.3 units and that the combination of the two anticarcinogens used produced a significant prolongation of the animals survival time with anticarcinogenic potency of 22.1 and 22.2 units, respectively. This is considered a potent anticarcinogenic effect. The question of an additional supportive administration of such agents complementary to the conventional cancer chemotherapy in humans is raised. Of course, further studies are needed.     

Effect of vitamins A, C and glutathione on the mutagenicity of benzo[a]pyrene mediated by S9 from vitamin A-deficient rats

Vitamin A deficiency has been shown to enhance the mutagenicity of benzo[a]pyrene (Narbonne et al., 1985). Here we report that this is not a result of increased benzo[a]pyrene metabolism but might be a consequence of either a lack of vitamin A or a decreased level of scavengers (ascorbic acid and glutathione) in the liver. However, the addition of vitamin A in vitro in the form of retinyl palmitate strongly inhibits the benzo[a]pyrene mutagenicity. An enhancing effect on the mutagenicity of benzo[a]pyrene is observed with addition of ascorbic acid when incubated with high amounts of the precarcinogen. In vivo addition of high levels of glutathione also reduces the mutagenicity of benzo[a]pyrene.     

Sulforaphane and its analogues inhibit CYP1A1 and CYP1A2 activity induced by benzo[a]pyrene

CYP1A1 and CYP1A2 enzymes metabolize polycyclic aromatic hydrocarbons (PAHs) to the reactive oxyderivatives. PAHs can induce the activity of both enzymes, which increases its conversion and enhances risk of carcinogenesis. Thus, the inhibition of CYP enzymes is recognized as a cancer chemoprevention strategy. A well‐known group of chemopreventive agents is isothiocyanates, which occur naturally in Brassica vegetables. In this paper, a naturally occurring sulforaphane and its two synthetic analogues isothiocyanate‐2‐oxohexyl and alyssin were investigated. The aim of the study was to determine whether the differences in the isothiocyanate structure change its ability to inhibit CYP1A1 and CYP1A2 activity induced by benzo[a]pyrene in HepG2 and Mcf7 cells. Also a mechanistic study was performed including isothiocyanates' influence on CYP1A1 and CYP1A2 catalytic activity, enzymatic protein level, and AhR translocation. It was shown that both enzymes were significantly induced by benzo[a]pyrene, and isothiocyanates were capable of decreasing the induced activity. The inhibitory properties depend on the types of isothiocyanate and enzyme. In general, CYP1A2 was altered in the more meaningful way than CYP1A1 by isothiocyanates. Sulforaphane exhibited weak inhibitory properties, whereas both analogues were capable of inhibiting BaP‐induced activity with the similar efficacy. The mechanistic study revealed that analogues decreased the CYP1A2 activity via the protein‐level reduction and CYP1A1 directly. The results indicate that isothiocyanates can be considered as potent chemopreventive substances and the change in the sulforaphane structure increases its chemopreventive potency. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:18–28, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20259     

Effects of vitamin A and insulin on the antioxidative state of diabetic rat heart: a comparison study with combination treatment

Because elevated oxidative stress may exacerbate cardiovascular complications of diabetes mellitus, the current study aimed to investigate the effects of treatment with either vitamin A, an antioxidant, or with insulin on lipid peroxidation products and antioxidant enzyme activities of diabetic rat heart. Also to evaluate whether a combination of vitamin A and insulin exerts more beneficial effects than treatment with each agent alone. Rats were made diabetic with a single injection of streptozotocin (STZ, 55 mg kg(-1) i.p.). Two days after STZ-injection, one group of diabetic rats was treated with vitamin A (retinol acetate, 30 mg kg(-1) day(-1) i.o.) for 12 weeks. A second group of diabetic rats was untreated for 6 weeks and then treated for another 6 weeks with insulin (8-10 IU rat(-1) day(-1) s.c.). Both therapies were applied to another group of diabetic rats for assessment of combined therapy with vitamin A plus insulin. Hearts from 12-week untreated diabetic animals showed about a four-fold increase in the level of thiobarbituric acid reactive substances (TBARS), indicative of increased lipid peroxidation. This was accompanied by approximately 100% increase in both catalase and glutathione peroxidase (GSHPx) enzyme activities. Therapy with insulin alone caused a small but significant improvement in plasma TBARS as well as GSHPx activities, but no significant change in plasma catalase in diabetic animals. Diabetes-induced disturbance in TBARS was almost completely prevented by vitamin A therapy. Although, a similar degree of activities for GSHPx was determined in diabetic animals treated with each agent alone, combination therapy was found to be more effective than single therapies in the recovery of GSHPx of diabetic heart. In contrast to insulin single therapy, vitamin A alone significantly prevented an increase in catalase activity of diabetic heart, and a combination of these agents did not supply any further benefit. Superoxide dismutase (SOD) activity was not found significantly different among the experimental groups. STZ-diabetes also resulted in less plasma retinol and retinol-binding protein (RBP), which was significantly improved by insulin single therapy while vitamin A used alone, failed to increase plasma retinol and RBP levels of diabetic animals. Our findings suggest that single therapy with insulin is unable to preclude oxidative reactions in diabetic heart to the same extent as obtained by vitamin A therapy alone, in spite of allowing recovery of normal growth rate and improved vitamin A metabolism in diabetic rats. A combination of insulin with vitamin A may provide more benefits than use of either agent alone in the treatment of general characteristics of diabetes and the maintenance of antioxidant defence of diabetic heart and thus in the reduction of peroxidative stress-induced cardiac injury.     

Effect of vitamin A deficiency on pulmonary and hepatic in vitro metabolism of benzo(a)pyrene in rat

The metabolism of (3H)-benzo(a)pyrene and the activities of enzymes involved in its metabolism were studied in rat lung and liver in vitamin A deficiency. Deficiency of vitamin A resulted a significant decrease in the overall metabolism of benzo(a)pyrene in the liver in vitro, whereas no significant difference was evident in the lung. The ethyl acetate-soluble metabolites of benzo(a)pyrene formed by lung and liver preparations were unaltered qualitatively by vitamin A deficiency. However, quantitative analysis revealed that vitamin A deficiency decreased the yield of dihydrodiols, quinones and phenols in liver, and dihydrodiols in lung. The hepatic cytochrome P-450 content, arylhydrocarbon hydroxylase and uridine diphosphate-glucuronosyl transferase activities were reduced, whereas glutathione S-transferase activity was increased in the vitamin A deficient animals. Contrary to this, pulmonary cytochrome P-450 content was above the control values (p less than 0.01) and no alteration in pulmonary arylhydrocarbon hydroxylase activity was observed in vitamin A deficient rats. Uridine diphosphate-glucuronosyltransferase and glutathione S-transferase activities were impaired in lung by inducing vitamin A deficiency. However, no significant difference was evident in the overall metabolism of benzo(a)pyrene by lung supernatants from the two groups.     

Effect of vitamin A deficiency on the pulmonary and hepatic drug-metabolizing enzymes in rat

The effect of vitamin A deficiency on the drug-metabolizing enzyme system of the lung and liver was analyzed in rats fed diets with or without vitamin A for 5-6 weeks. The hepatic level of vitamin A was significantly reduced in vitamin A deficient animals. The hepatic cytochrome P-450 and b5 contents and activity of benzo(a)pyrene hydroxylase was significantly reduced in deficient animals. Contrary to this, pulmonary cytochrome P-450 and b5 contents were above the control values. No alteration in pulmonary benzo(a)pyrene hydroxylase was noted. The uridine diphosphate-glucuronosyltransferase activity of digitonin-treated microsomal membranes was below the control values both in lung and liver. However, the native uridine diphosphate-glucuronosyltransferase activity remained unchanged in the liver and was below control values in the lung.     

Hepatic uptake and antihepatotoxic properties of vitamin E and liposomes in the mouse

Intravenous administration of soybean phosphatidylcholine liposomes containing different amounts of tocopherol acetate leads to a dose and time dependent increase of mouse liver tocopherol content, which was not observed when the preparation was given orally. When benzo[a]pyrene pretreated mice intoxicated with 400 mg/kg AAP were pretreated 2 h before with 1 g/kg phosphatidylcholine liposomes containing 4 mg/kg vitamin E acetate, these animals were protected against liver damage. Vitamin E alone or liposomes lacking vitamin E showed no protection. In an inflammatory liver disease model, i.e. fulminant hepatitis induced by intraperitoneal administration of 700 mg/kg galactosamine and 1 microgram/kg lipopolysaccharide phosphatidylcholine liposomes protected at a dose of 1 g/kg i.v. In this case, however, the protection was not due to the presence of vitamin E. These findings demonstrate the usefulness of phosphatidylcholine for liver protection and show that the protective spectrum is improved when they contain vitamin E. The data suggest that phosphatidylcholine is an excellent carrier for delivery of vitamin E to the liver.     

The homogeneity of rat liver microsomal cytochrome P-448 activity and its role in the activation of benzo[a]pyrene to mutagens

The O-deethylation of ethoxyresorufin and the metabolic activation of benzo[a]pyrene to mutagens were determined in hepatic microsomal preparations from control and induced animals. An excellent direct correlation (r = 0.95) has been observed between ethoxyresorufin O-deethylase and the metabolic activation of benzo[a]pyrene to mutagens when the fraction of cytochromes P-450 present as cytochrome P-448 was altered by the administration of phenobarbitone and 3-methylcholanthrene alone or in combination with 9-hydroxyellipticine. The correlation between these activities was maintained following treatment of animals with Arochlor 1254, benzo[a]pyrene, benzo[e]pyrene, 7,12-dimethylbenzo[a]anthracene,2-anthramine and 2-naphthylamine.     

Effect of intratracheally instilled benzo(a)pyrene on the pulmonary and hepatic drug-metabolizing enzymes in normal and vitamin A deficient rats

The effect of intratracheal instillation of different doses of benzo(a)pyrene (0.1, 1.0 and 2.0 mg) on the drug metabolizing enzymes of lung and liver was analysed in rats fed diet with or without vitamin A for 5-6 weeks. Benzo(a)pyrene exposure at 2.0 mg dose only elevated the level of cytochrome P-450 and b5, and activity of benzopyrene hydroxylase in liver, and extent of increase was similar in normal and vitamin A deficient groups. Contrary to this, pulmonary contents of cytochrome P-450 and b5, and benzopyrene hydroxylase activity increased over control values in both the groups even at lower doses of benzo(a)pyrene. Moreover, their values were higher in vitamin A deficient-treated groups compared to normal-treated controls. Increase in these parameters was greater in lung as compared to increase in liver. NADPH cytochrome C-reductase in lung and liver was not affected either by inducing vitamin A deficiency or exposing these rats further to benzo(a)pyrene. Uridine-diphospho-glucuronosyl-transferase (UDP-GT) activity in normal and vitamin A deficient groups was enhanced following exposure to benzo(a)pyrene both in lung and liver. However, activity of this enzyme remained impaired in vitamin A deficient groups, benzo(a)pyrene exposed or not exposed when compared to respective normal controls. Glutathione S-transferase activity remained unchanged following exposure to benzo(a)pyrene both in lung and liver. The apparent increase in hepatic glutathione S-transferase and decrease in pulmonary glutathione S-transferase activity in vitamin A deficiency was only due to vitamin A deficient status of rats with no further effect of benzo(a)pyrene.     

Effects of the antioxidants curcumin and vitamin C on cisplatin-induced clastogenesis in Wistar rat bone marrow cells

The use of dietary antioxidants to prevent antitumor agent-induced chromosomal damage in nontumor cells is currently eliciting considerable interest. Curcumin (CMN) is a dietary antioxidant that has been reported to protect against clastogenesis in in vivo and in vitro assays. This study was undertaken to investigate the modulatory effects of CMN on cisplatin-induced chromosomal aberrations in Wistar rat bone marrow cells and whether there is any potentiation of these effects with the combination between CMN and vitamin C (VC), which has been reported to reduce the clastogenic effect of many antitumor agents in in vivo assays. Animals treated with CMN plus a single dose of cisplatin, at 18, 24 or 72 h following treatment, presented a statistically significant reduction in the total amount of chromosomal damage and in the number of abnormal metaphases. The results also indicate that the combination between antioxidants would not be effective in protecting against cisplatin-induced chromosomal damage in animals sacrificed 24 h after cisplatin treatment. Under the present experimental conditions, CMN could prevent cisplatin-induced clastogenesis by acting as a free radical scavenger.     

Benzo(a) pyrene metabolism in vitamin A deficient rats

Hepatic microsomal metabolism of benzo(a)pyrene, a representative carcinogenic polycyclic hydrocarbon and an ubiquitous environmental pollutant was studied in control and vitamin A deprived (10–12 weeks) male rats. Hydroxylation of benzo(a)pyrene to fluorescent phenols was found to be significantly depressed in the deficient animals. The decreased hepatic metabolism may lead to delayed clearance of the carcinogenic chemicals in this condition and thus may explain at least in part the enhanced susceptibility to carcinogenesis in hypovitaminosis A.     

Effect of vitamin C and vitamin E on prostaglandin synthesis by fibroblasts and squamous carcinoma cells.

Dietary levels of vitamins C and E have been associated with cancer prevention and to a lesser extent with therapeutic enhancement of cancer treatment. Inhibition of prostaglandins (PGs) by pharmacological agents has been demonstrated to enhance immunocompetence, and to suppress growth of tumors in animals and humans. We report here on the effect of vitamins C and E on PGE2 production by human gingival fibroblasts and SCC-25 oral squamous carcinoma cells. The results indicate: 1. vitamins C and E exert a dose-dependent effect on arachidonic acid (AA) release and PGE2 synthesis; 2. vitamin E has a biphasic effect which is stimulatory at 1 and 10 microM and inhibitory at 100 microM; 3. vitamin E is considerably more potent than vitamin C in its inhibitory effect on AA and PGE2 in both cell types; 4. a combination of the two vitamins has a consistent dose-dependent inhibitory effect on AA and PGE2; 5. vitamin C stimulates PGE2 synthesis from exogenous AA in fibroblasts, and inhibits it in SCC-25 cells. The in vivo significance of these findings requires further investigation.     

Modulation of the effects of tumor therapeutic agents by vitamin C

In vitro and in vivo results suggest that sodium ascorbate (vitamin C) could be useful in the management of neoplasms provided it is used on a biological rationale. Vitamin C can exert multiple mechanisms of action depending upon the cell type and experimental conditions. Vitamin C may kill certain tumor cells, may increase the cell killing effect of certain tumor therapeutic agents and may stimulate the host's immune system against the residual tumor cells. In vitro data also suggest that the irrational use of vitamin C in the management of neoplasms could be ineffective and even harmful. Further study on the effects of vitamin C in combination with tumor therapeutic agents must be done, using animal tumor models before assaying its role in the management of human neoplasms.     

Effects of dietary benzo(a) pyrene on intestinal phase I and phase II drug metabolizing systems in normal and vitamin A-deficient rats

Feeding of vitamin A-deficient diet to male weanling rats for 10 weeks caused significant increase in the activities of Phase I enzyme system, i.e., cytochrome P-450, cytochrome b5 and arylhydrocarbon hydroxylase in the proximal, middle and distal segments of the intestine. Of the Phase II enzymes studied, UDP-glucuronyltransferase showed significant decrease whereas glutathione S-transferase showed significant increase. Treatment with benzo(a)pyrene caused greater induction in the levels of Phase I enzymes in deficient animals as compared to controls. In contrast to this, benzo(a)pyrene treatment induced the level of UDP-glucuronyltransferase in control rats more than in deficient rats. Intestinal NADPH cytochrome C-reductase and glutathione S-transferase remained insensitive to benzo(a)pyrene induction.     

Paradoxical effects of vitamin C in Chagas disease

Trypanosoma cruzi infection stimulates inflammatory mediators which cause oxidative stress, and the use of antioxidants can minimize the sequelae of Chagas disease. In order to evaluate the efficacy of vitamin C in minimizing oxidative damage in Chagas disease, we orally administered ascorbic acid to Swiss mice infected with 5.0?×?10⁴ trypomastigote forms of T. cruzi QM2 strain. These animals were treated for 60?days to investigate the acute phase and 180?days for the chronic phase. During the acute phase, the animals in the infected and treated groups demonstrated lower parasitemia and inflammatory processes were seen in more mice in these groups, probably due to the higher concentration of nitric oxide, which led to the formation of peroxynitrite. The decrease in reduced glutathione concentration in this group showed a circulating oxidant state, and this antioxidant was used to regenerate vitamin C. During the chronic phase, the animals in the infected and treated group showed a decrease in ferric reducing ability of plasma and uric acid concentrations as well as mobilization of bilirubin (which had higher plasma concentration), demonstrating cooperation between endogenous non-enzymatic antioxidants to combat increased oxidative stress. However, lower ferrous oxidation in xylenol orange concentrations was found in the infected and treated group, suggesting that vitamin C provided biological protection by clearing the peroxynitrite, attenuating the chronic inflammatory process in the tissues and favoring greater survival in these animals. Complex interactions were observed between the antioxidant systems of the host and parasite, with paradoxical actions of vitamin C.     

Regeneration of static-load-degenerated articular cartilage extracellular matrix by vitamin C supplementation

The effect of a physiological dose of vitamin C (100 mug/ml) on goat articular cartilage chondrocytes cultured in an alginate matrix and subjected to static pressurization of 2.4 MPa was investigated. Biochemical analyses of DNA, glycosaminoglycan (GAG), collagen and protease activity were carried out in various matrix fractions, i.e. cellular matrix (CM) and further removed matrix (FRM), and in culture medium. The treatment of chondrocytes with vitamin C after static pressure increased the GAG content in both CM and FRM (P < 0.03) as compared with control or vitamin C/ static load alone. The collagen content of chondrocytes treated with vitamin C alone and vitamin C after static load also increased significantly in FRM (P < 0.003) as compared with control and static load alone. The specific activity of protease in CM and FRM decreased after vitamin C supplementation both with and without static pressure relative to control (P < 0.003). Transmission electron-microscopic images showed a mixed population of spherical and elliptical chondrocytes when vitamin C was added after static load as compared with static load alone where only elliptical cells were seen. Abundant pericellular and collagen fibrils were seen in this group of chondrocytes as compared with all other groups and the control. The results thus show that, in vitro, vitamin C supplementation of chondrocytes after static loading has the potential to reduce the morphological and biochemical degeneration of chondrocytes caused by static loading, thereby improving the cellular health and functioning of articular cartilage.     

Enhancement of antitumor effect of paclitaxel in combination with immunomodulatory Withania somnifera on benzo(a)pyrene induced experimental lung cancer

The current experimental work deals with the immunomodulatory studies on the extract of Withania somnifera (L.) Dunal root powder against benzo(a)pyrene induced lung cancer in male Swiss albino mice. In our previous study, we reported the antioxidant and anticarcinogenic effect of W. somnifera (L.) Dunal along with paclitaxel. Immune dysfunction has been found to be associated with cancer and chemotherapy. Benzo(a)pyrene induced cancer animals were treated with 400mg/kg bodyweight of W. somnifera (L.) Dunal extract for 30 days significantly alters the levels of immunocompetent cells, immune complexes and immunoglobulins. Based on the data, the carcinogen as well as the paclitaxel affects the immune system, the toxic side effects on the immune system is more reversible and more controllable by W. somnifera (L.) Dunal. These results concluded the immunomodulatory activity of W. somnifera (L.) Dunal extract, which is a known immunomodulator in indigenous medicine.     

Enhancement and inhibition of benzo[a]pyrene-induced SOS function in E. coli by synthetic antioxidants

8 antioxidants were tested in the SOS chromotest for induction of SOS function and for modulation of benzo[a]pyrene-induced SOS function. None of the antioxidants leads to increased beta-galactosidase activity by itself. Butylated hydroxytoluene at concentrations between 10(-5) M and 3 X 10(-4) M enhances benzo[a]pyrene-induced SOS function at benzo[a]pyrene concentrations between 10(-6) M and 3 X 10(-5) M. Butylated hydroxyanisole, ethoxyquin, propyl gallate and octyl gallate also slightly enhance benzo[a]pyrene-induced SOS function at concentrations up to 3 X 10(-4) M though to a lesser degree than butylated hydroxytoluene. Dodecyl gallate, vitamin C and alpha-tocopherol do not increase benzo[a]pyrene action. In concentrations exceeding 3 X 10(-4) M all synthetic antioxidants tested but not vitamin C and alpha-tocopherol decrease beta-galactosidase activity both in the absence and, more extensively, in the presence of benzo[a]pyrene. Preliminary data suggest that the apparent suppression of benzo[a]pyrene-induced SOS function is not due to an effect on the formation of benzo[a]pyrene metabolites by the metabolizing system used.     

Modulation of aortic protein phosphorylation by benzo(a)pyrene: Implications in PAH-induced atherogenesis

The toxicity of polycyclic aromatic hydrocarbons such as benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, and 3-methylcholanthrene has been associated with alterations in the proliferation of vascular smooth muscle cells and the development of lesions of mesenchymal origin. Because phosphorylation of endogenous substrates plays a central role in the regulation of smooth muscle cell growth, the present studies were conducted to evaluate the phosphorylation pattern of medial aortic protein upon repeated in vivo exposure of Japanese quail to benzo(a)pyrene (BaP). Medial aortic homogenates from quail treated for 10 weeks with 10 mg/kg benzo(a)pyrene or vehicle were processed for in vitro measurements of protein phosphorylation. In vitro phosphorylation of endogenous or exogenous proteins stimulated in vitro by phorbol myristate acetate/phosphatidyl-serine or cyclic AMP, known activators of protein kinase C and cyclic AMP-dependent protein kinase, respectively, was examined in the cytosolic and particulate fractions of homogenates from control and treated animals. Benzo(a)pyrene treatment significantly enhanced the basal phosphorylation of Mr 113, 35, and 23 kDa proteins in the cytosolic fraction. Modest increases in the phosphorylation of Mr 71, 52, and 38 kDa were also observed under basal conditions. No changes in the basal phosphorylation of particulate proteins were observed. Phosphorylation of endogenous protein substrates by protein kinase C in the cytosolic fraction was not altered by benzo(a)pyrene treatment. In contrast, inhibition of C-kinase-mediated phosphorylation of endogenous Mr 272, 72, and 45 kDa proteins was observed in the particulate fraction of aortic homogenates from benzo(a)pyrene-treated quail relative to controls. Exogenous histone phosphorylation by PKC in the particulate, but not cytosolic fraction, was decreased by benzo(a)pyrene treatment. The effects of benzo(a)pyrene on the C-kinase system were specific, since cAMP-mediated phosphorylation of endogenous proteins, as well as exogenous histone, was not altered by benzo(a)pyrene. Interestingly, benzo(a)pyrene treatment was associated with a selective increase of Mr 200, 80, and 67 kDa proteins in the cytosolic fraction. Collectively, these data are consistent with the hypothesis that medial protein phosphorylation is a significant molecular target of benzo(a)pyrene within the vascular wall.     

Prostaglandin synthetase dependent benzo(a)pyrene oxidation: products of the oxication and inhibition of their formation by antioxidants.

The products of the arachidonic acid dependent oxidation of benzo(a)pyrene by enzyme preparations from sheep seminal vesicles are the 1,6?, 3,6?, and 6,12? quinones. The metabolites were identified by high performance liquid chromatography and visible spectroscopy. The amount of benzo(a)pyrene converted to quinones by a Tween 20 solubilized preparation during a 15 min period is 43 μM/mg protein. The relative yields of the individual quinones are 1,6 – 25%, 3,6 – 30%, and 6,12 – 45%. Arachidonate dependent benzo(a)pyrene oxidation is strongly inhibited by butylated hydroxyanisole, vitamin E, and diethyldithiocarbamate and moderately inhibited by butylated hydroxytoluene and vitamin C. Epinephrine and lipoic acid are also inhibitors.