3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induces caspase-dependent apoptosis in mononuclear cells.

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), one of the tryptophan pyrolysates, is a dietary carcinogen and is formed in cooked meat and fish in our daily diet. Trp-P-1 will affect the cells in the blood circulation system before it causes carcinogenicity in target organs such as the liver. In this study, the cytotoxicity of Trp-P-1 was investigated in mononuclear cells (MNCs) from blood. Trp-P-1 (10-15 microM) decreased cell viability and induced apoptosis characterized both by morphological changes and by DNA fragmentation 4 h after treatment. DNA fragmentation was also observed following treatment at 1 nM after 24 h in culture. This result suggested that apoptosis would occur in the body following unexpected intake of foods containing Trp-P-1. To determine the mechanism of apoptosis, we investigated the activation of the caspase cascade in MNCs. Trp-P-1 (10-15 microM) activated the caspase cascade, i.e. the activity of caspase-3, -6, -7, -8 and -9 increased dose-dependently using peptide substrates, the active forms of caspase-3, -8 and -9 were detected by immunoblotting, and cleavage of poly(ADP-ribose) polymerase and protein kinase C-delta as the intracellular substrates for caspases was observed. A peptide inhibitor of caspase-8 completely suppressed activation of all other caspases, while an inhibitor of caspase-9 did not. These results indicated that caspase-8 may act as an apical caspase in the Trp-P-1-activated cascade.

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induces apoptosis in rat splenocytes and thymocytes by different mechanisms.

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is a potent carcinogen present in cooked meat. Although the target of this carcinogen is mainly in the liver, Trp-P-1 is distributed in the thymus and spleen as well as in the liver after administration. However, the cytotoxic effect of Trp-P-1 on lymphocytes has not been examined in detail. In the present study, we investigated the cytotoxicity of Trp-P-1 against rat splenocytes and thymocytes. Trp-P-1 reduced viability of both types of cells in the same manner, the LD(50) at 6h in culture was 15 microM, and the time for the 50% decrease in cell viability (t(1/2)) at 20 microM was 3h. In both types of cells, Trp-P-1 caused the activation of caspase-3-like proteases and the cleavage of poly(ADP-ribose) polymerase, both of which are biochemical markers of apoptosis. On the other hand, DNA fragmentation occurred in splenocytes, but not in thymocytes although Trp-P-1 activated 32-34kDa nucleases that may not be able to degrade DNA into nucleosomal units. These results indicated that Trp-P-1 induces apoptosis in both splenocytes and thymocytes by different mechanisms in which distinct apoptotic pathways may exist downstream of the caspase cascade.

Detection of biomarkers for apoptosis in rat liver after perfusion with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1).

We previously demonstrated that 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induced apoptosis in primary cultured rat hepatocytes. In this study, we investigated apoptotic biomarkers in rat liver after perfusion with 30 microM Trp-P-1 as preliminary experiments for in vivo study. Induction of c-Myc and p53 protein and the activities of caspase-3, -6, and -8 were detected in Trp-P-1-perfused liver. In addition, Trp-P-1 modulated the DNA binding activity of the apoptosis-related transcription factors, NF-kappaB and AP-1. These results imply a possibility that Trp-P-1 would induce apoptosis in vivo.

Synergistic effects of food colors on the toxicity of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in primary cultured rat hepatocytes.

The synergistic effect of food additives or food colors on the toxicity of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was investigated using primary cultured rat hepatocytes. When hepatocytes from rats fed a standard diet were treated with a mixture of four major food additives (sorbitol, sodium L(+)-glutamate, benzoic acid, and propylene glycol) or a mixture of six typical artificial food colors (erythrosine, allura red, new coccine, brilliant blue, tertrazine, and fast green), the in vitro treated food-color mixture itself showed cytotoxicity: the reduction of cell viability and decreases in the activities of gluconeogenesis and ureogenesis. The food-color mixture enhanced cytotoxicity of Trp-P-1 obviously. We then investigated the effects of in vivo-dosed food additives or food colors on Trp-P-1-caused toxicity. Hepatocytes were isolated and cultured from rats fed a diet containing a mixture of food additives or a mixture of food colors with half the amount of their respective acceptable daily intake for 4 wk. Trp-P-1 was administered to the hepatocytes at various concentrations for 12 h. Synergistic effects of in vivo-dosed food additives and food colors were not observed on Trp-P-1-caused cytotoxicity as estimated by a loss of cell viability and the reductions of DNA and protein syntheses. On the contrary, we have observed that in vivo administered food colors synergistically facilitated to reduce the activities of gluconeogenesis and ureogenesis in Trp-P-1-treated hepatocytes. These results suggest that the daily intake of artificial food colors may impair hepatic functions such as gluconeogenesis and ureogenesis, when dietary carcinogens are exposed to the liver cells.

Cytochrome P4501A1-inhibitory action of antimutagenic anthraquinones in medicinal plants and the structure-activity relationship.

We have earlier found that flavones and flavonols in vegetables specifically inhibited one of the carcinogenesis-related enzymes, cytochrome P450 (CYP) 1A1, and subsequently suppressed the mutagenicity of food-derived carcinogens. In this study, we explored other candidates for the enzyme inhibitor in Chinese medicinal plants. Some of them were antimutagenic toward 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). For example, Rheum officinale contained large amounts of anthraquinones as the active compounds, 3.4 mg of emodin, 2.1 mg of chrysophanol and 1.8 mg of rhein in 10 g of dry matter. Anthraquinones showed similar IC50 values for antimutagenicity against Trp-P-2 to those for inhibition of the N-hydroxylation activity of CYP1A1 toward Trp-P-2, indicating that the antimutagenicity was attributable to CYP inhibition. The structure-activity relationships were then examined with 14 commercial chemicals, and it was found that the interaction with an enzyme required three rings and an oxygen group in the side ring. This characteristic is similar to that of flavones and flavonols.

Dietary antioxidants fail in protection against oxidative genetic damage in in vitro evaluation.

Carcinogenesis is believed to be induced through the oxidative damage of DNA, and antioxidants are expected to suppress it. So, the polyphenolic antioxidants in daily foods were investigated to see whether they protect against genetic damage by active oxygen. In the evaluation, we used a bioassay and a chemical determination, a Salmonella mutagenicity test for mutation by a N-hydroxyl radical from one of the dietary carcinogens 3-amino-1-methyl-5H-pyrido[4,3-b]indole and the formation of 8-hydroxyl (8-OHdG) from 2'-deoxyguanosine (2'-dG) in a Fenton OH-radical generating system. Thirty-one antioxidants including flavonoids were compared in terms of radical-trapping activity with bacterial DNA and 2'-dG. Antioxidants inhibited the mutation but the IC50 values were in the mM order. Against 8-OHdG formation, only alpha-tocopherol had a suppressive effect with an IC50 of 1.5 microM. Thus, except alpha-tocopherol, the dietary antioxidants did not scavenge the biological radicals faster than bacterial DNA and intact 2'-dG, indicating that they failed to prevent oxidative gene damage and probably carcinogenesis.

Comparison in metabolic activity of cytochrome P450 1A1 on heterocyclic amines between human and rat.

In mutagenicity and antimutagenicity tests, the toxicants have been activated to the ultimate mutagenic forms usually with rat cytochrome P450 (CYP) enzymes. An understanding is important of whether these data can be available for human. In this paper are compared the activating abilities of CYP1A1 between human and rat using recombinant yeast cells that express respective CYP1A1 and yeast NADPH-CYP-oxidoreductase simultaneously. Three different types of dietary procarcinogens, heterocyclic amines, were tested by two methods: a bioassay with Salmonella mutagenicity test and a chemical determination of N-hydroxyls as the ultimate mutagenic forms. Compared with ED(50) values, saturation levels, and V(max)/K(m) values at an initial stage of the enzyme activity, human and rat CYP1A1 showed almost similar abilities for the metabolic activation on heterocyclic amines. The two enzymes also had the same preference for the tested procarcinogens and the same affinities to the specific inhibitors such as flavonoids.

Antimutagenicity of flavones and flavonols to heterocyclic amines by specific and strong inhibition of the cytochrome P450 1A family.

We found the mechanism in flavonoids that can strongly suppress the mutagenicity of one of the food-derived and carcinogenic heterocyclic amines, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The antimutagenicity was evaluated by IC50 value, the amount required for 50% inhibition of the mutagenicity of 0.1 nmol Trp-P-2, with Salmonella typhimurium TA98 strain in the presence of S9 mix. The flavones and flavonols were two orders stronger as antimutagens that such antimutagenic phytochemicals as chlorophylls and catechins. We had previously found flavonoids to be a desmutagen to neutralize Trp-P-2 before or during attack of DNA, because they had no effect on either the ultimate mutagenic form of Trp-P-2 (N-hydroxy-Trp-P-2) or the mutated cells. The desmutagenicity of the flavonoids did not depend on the hydroxy number or position that should be associated with antioxidative potency, and was also unaffected by the solubility of Trp-P-2 in the assay solution. The inhibitory effect of the flavonoids on the metabolic activation of Trp-P-2 to N-hydroxy-Trp-P-2 was almost in parallel with the antimutagenic IC50 value, when determined with a Saccharomyces cerevisiae AH22 cell simultaneously expressing both rat cytochrome P450 1A1 and yeast reductase. The Ki values of flavones and flavonols for the enzyme were less than 1 microM, while the K(m) value of Trp-P-2 was 25 microM. The antimutagenicity of the flavones and flavonols was thus concluded to be due to inhibition of the activation process of Trp-P-2 by P450 1A1 to the ultimate carcinogenic form. They were also able to act as antimutagens toward other indirect mutagens that were activated by P450 1A1.

Xenobiotic tolerance of primary cultured hepatocytes in rats fed a high-fat or high-protein diet.

The diet and nutritional status dominate a tolerance to environmental xenobiotics. In this study, the cytotoxic action of carbon tetrachloride (CCl4) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), one of the dietary carcinogens, was investigated using primary cultured hepatocytes from rats fed a high-fat (23% corn oil) or high-protein (50% casein) diet for three weeks. Both chemicals showed strong cytotoxicity to hepatocytes, which was judged by measurement with the MTT-test and lactate dehydrogenase leakage test. A dietary effect on cytotoxicity was observed; hepatocytes from rats fed the high-protein diet were more susceptible to cytotoxicity than the cells from rats fed a standard diet. On the other hand, ureogenesis, as a cellular function of hepatocytes, was markedly decreased in the cells from rats fed the high-fat diet. These activities were affected in the CCl4-treated cells but not in the Trp-P-1-treated cells. The same trend of both diet and chemical effects was observed in gluconeogenesis from fructose. We conclude that the hepatocytes from rats fed a high-protein diet have high susceptibility to the cytotoxicity of CCl4 and Trp-P-1, but cytotoxicity was not related to the reduction of cellular functions.

Tryptophan pyrolysis products, Trp-P-1 and Trp-P-2 induce apoptosis in primary cultured rat hepatocytes.

The cytotoxicity of heterocyclic amines, dietary carcinogens derived from cooked foods, to primary cultured rat hepatocytes was studied. A tryptophan pyrolysis product, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was the most cytotoxic of 11 compounds tested. Trp-P-1 was found to induce apoptosis as measured by morphological changes in nuclear chromatin and internucleosomal DNA fragmentation. 3-Amino-1-methyl-5H-pyrido[4,3-b] indole (Trp-P-2) showed a moderate apoptotic effect, and other compounds had a similar but weaker effect.

Three-dimensional structure of maize alpha-zein proteins studied by small-angle X-ray scattering.

alpha-zeins of maize (Zea mays) that are storage proteins contain nine or ten tandem repeats comprising of about 20 amino acids. Small-angle X-ray scattering (SAXS) of alpha-zeins was measured in 70% (v/v) aqueous ethanol containing beta-mercaptoethanol or without reagent in a protein concentration range of 2.0 to 40.0 mg/ml. The overall radius of gyration of whole particles, Rg, and the corresponding radius of gyration of the cross-section, Rc, of reduced alpha-zeins are 4.00 +/- 0.03 nm and 1.39 +/- 0.05 nm, respectively, in the 70% (v/v) aqueous ethanol containing 2% (v/v) beta-mercaptoethanol. Analyses using the Rg and Rc values indicate that reduced alpha-zeins exist as asymmetric particles with the length of about 13 nm in the solution. A structural model is developed under assumption that each of tandem repeats units forms single alpha-helix and they are joined by glutamine-rich 'turns' or loops, as employed by Argos et al., [Argos, O., Pedersen, K., Marks, M.D. and Larkins, B.A. (1982) J. Biol. Chem. 257, 9984-9990] and Garratt et al. [Garratt, R., Oliva, G., Caracelli, I., Leite, A. and Arruda, P. (1993) Proteins Struc. Func. Genet. 15, 88-99], and that the longest dimension of 13 nm comes from linear stacking of the anti-parallel helices of tandem repeat in the direction perpendicular to the helical axis. The resultant model is presented by an elongated prism-like shape with an approximate axial ratio of 6:1.

Effects of dietary lipid peroxidation products on hormonal responses in primary cultured hepatocytes of rats.

Dietary lipid peroxidation products cause endogenous lipid peroxidation with hepatic dysfunction. In this study, we isolated and cultured hepatocytes of rats that were given secondary autoxidation products of linoleic acid (p.o., 400 mg/rat/day for 3 days), and examined the hormonal responses of these hepatocytes. An increase in thiobarbituric acid reactive substances and a depletion of vitamin E persisted in hepatocytes from treated rats for at least 24 h in culture as compared to those from control rats. As markers for hepatic dysfunction, the activities of six enzymes were measured. In each case, there was an initial decrease in the enzyme activity in hepatocytes from the treated rats, and all activities were restored by 48 h in culture. Then, we measured the hormonal responses of these hepatocytes. The responses to insulin or glucagon in hepatocytes from secondary products-treated and control rats were the same. In contrast, the response to dexamethasone was significantly lowered in hepatocytes from secondary products-treated rats as measured by the induction of tryptophan 2,3-dioxygenase and tyrosine aminotransferase. We conclude that primary cultured hepatocytes from the rats treated in vivo with dietary lipid peroxidation products retained symptoms of oxidative stress and had a low response to glucocorticoids.

Hepatic Phosphoglucomutase Activity as a Marker of Oxidative Stress Induced by Pro-oxidative Drugs.

A decrease in hepatic phosphoglucomutase activity was found after intraperitoneal doses to rats with pro-oxidative drugs; carbon tetrachloride, ethanol, paraquat, phenobarbital, thiopental, 3-methylcholanthrene, benzo[a]pyrene, Trp-P-1, benzene, and polychlorinated biphenyl. When the drugs were given at half of their LD50 values, except ethanol, hepatic phosphoglucomutase activities were decreased, and aldehydes which were detected by the thiobarbituric acid test were simultaneously accumulated 24 h after administration, in the liver. A significant correlation between phosphoglucomutase activity and the accumulations of aldehydes was obtained (r = - 0.536). No other changes in hepatic enzymes and metabolites were commonly observed. On the other hand, the low-molecular-weight aldehyde fraction in secondary products of linoleic acid decreased phosphoglucomutase activity in hepatocytes cultured with dexamethasone. It was concluded that aldehydes originating from endogenous lipid peroxidation decreased phosphoglucomutase activity in the liver, and the assay of hepatic phosphoglucomutase activity was useful as a marker of oxidative stress induced by pro-oxidative drugs.

Small-angle X-ray scattering study by synchrotron orbital radiation reveals that high molecular weight subunit of glutenin is a very anisotropic molecule.

Small-angle X-ray scattering of one high molecular weight (HMW) subunit of wheat glutenin was measured at protein concentration ranges from 1.0 to 10.0 mg/ml. The radius of gyration of whole particles, RO, in aq. 50% (v/v) 1-propanol and 0.1M acetic acid was 16.6 +/- 0.1nm and 22.8nm, respectively, and the corresponding radius of gyration of the cross-section, RC, was 2.82 +/- 0.02 nm and 2.23 +/- 0.01 nm, which indicate that the glutenin HMW subunit exists as very anisotropic particles in both solutions. The RO and RC values of the subunit, and the drastic decrease in scattered intensity at small angles that occurs in the acetic acid solution with relatively low protein concentration are completely explained in terms of rod-like molecules of the glutenin HMW subunit.

Mutagen formed from tryptophan reacted with sodium nitrite in acidic solution.

The reaction products from L-tryptophan treated with nitrite under acidic conditions were investigated for mutagenic activity with the Salmonella typhimurium his reversion assay and for DNA-damaging activity using the rec-assay. The diethyl ether extract of the reaction mixture showed 8 spots on thin-layer chromatography (TLC). One compound from the TLC had high mutagenic activity for TA98 without S9 mix, with little DNA-damaging activity. The mutagen was purified and identified by instrumental analysis as 2-hydroxy-(1-N-nitrosoindole)propionic acid (NIHP). The mutagenic activity of NIHP was determined by the induced mutation frequency method; the induced mutation frequency was about 19.2 X 10(-5) at a dose level of 160 micrograms/plate.

The effects of orally administered linoleic acid and its autoxidation products on intestinal mucosa in rat.

Linoleic acid and its autoxidation products, hydroperoxides and their secondary products, were orally administered to rats (350 mg each/rat). Hemorrhage was seen in the alimentary canal at 6 h after the dose of hydroperoxides. To examine their toxicities on intestinal mucosa, the activities of mucous enzymes (sucrase, maltase, and alkaline phosphatase) were measured. Hydroperoxides and their secondary products decreased the enzyme activities in jejunum at 6 h after the doses and increased them in both jejunum and ileum at 15 h, as compared to linoleic acid or saline solution. The decrease of enzyme activity was marked in the hydroperoxide group and the increase was marked in the secondary product group. Then, in in vitro experiments, the effects of autoxidation products on these enzymes were determined. Autoxidation products inactivated only alkaline phosphatase. Thus, the results in vivo disagreed with those in vitro. It was assumed that autoxidation products orally administered attacked a membrane of intestinal microvilli whereas in vitro they directly affected the enzymes.

Effect of orally administered secondary autoxidation products of linoleic acid on carbohydrate metabolism in rat liver.

The effects of orally administered secondary autoxidation products of linoleic acid in rat liver were investigated. Their administration led to two toxic effects on hepatic carbohydrate metabolism, as compared to the administration of saline or linoleic acid used as controls. One effect was depletion of glucose 6-phosphate and fructose 6-phosphate caused by the reduction of glycolysis and glycogenolysis, accompanied by decreases in glycogen synthesis and pentose phosphate cyclic activity. The reduction in these metabolic systems seems unlikely to occur because phosphofructokinase was regulated by ATP or citrate enzymatically, because their accumulation in the liver was not detected in the secondary products. Another toxic effect was the depletion of oxaloacetate and isocitrate caused by the reduction in enzyme activity of the mitochondrial citrate cycle. On the basis of these results, the hepatotoxic effects of secondary products are discussed as follows: the incorporated secondary products impaired the activities of hexokinase and phosphoglucomutase in the liver. The reduction in these enzyme activities resulted in the depletion of glucose 6-phosphate and fructose 6-phosphate, which led ultimately to decreases in the activities of phosphofructokinase, the pentose phosphate cycle, and glycogen synthesis. Moreover, the secondary products disturbed the mitochondrial membrane, resulting in a decrease in the activity of the citrate cycle, which was accompanied by depletion of its metabolites.

Mutagens formed from butylated hydroxyanisole treated with nitrite under acidic conditions.

The reaction products from butylated hydroxyanisole treated with nitrite under acidic conditions were investigated for mutagenic activity in Salmonella typhimurium his reversion assay and for DNA-damaging activity using H17 Rec+ (wild) and M45 Rec- (recombinationless) of Bacillus subtilis. The chloroform extract of the reaction mixture showed 9 spots on thin-layer chromatography (TLC). Compounds from 2 spots on the TLC had high mutagenic activity in TA100 without S9 mix, with DNA-damaging activity. The 2 mutagens were then crystallized from the reaction mixture and identified to be 2-tert.-butyl-p-quinone (t-BQ) and the dimer of t-BQ; 3,3'-di-tert.-butyl-biphenyldiquinone-(2,5,2',5') (BBDQ), from their instrumental analysis. The mutagenic activities of t-BQ and BBDQ were determined by Ames test, and the induced mutation frequencies were about 1.9 X 10(-4) (t-BQ) and 8.3 X 10(-5) (BBDQ).

Formation of DNA-damaging and mutagenic activity in the reaction systems containing nitrite and butylated hydroxyanisole, tryptophan, or cysteine.

It was confirmed by the procedure of rec-assay that DNA-damaging activities were formed in the reaction systems containing nitrite and phenol derivatives including BHA, tryptophan or cysteine under gastric pH conditions. The mutagenic action of the nitrite-BHA, nitrite-tryptophan and nitrite-cysteine systems was also tested according to Ames' method using Salmonella typhimurium TA 1535 and TA 98. The mutagenic activity was observed in the nitrite-tryptophan and nitrite-cysteine systems, though the nitrite-BHA system did not show the activity. The DNA-damaging products were generally labile, i.e., the activity decreased significantly after 1.5 to 2 hours of the reaction, except in the case of the nitrite-BHA system. The DNA-damaging activity in the nitrite-BHA system did not decrease even after 48 hours of the reaction. Nitrosophenol derivatives themselves showed the DNA-damaging activity at pH 1. The active product in the nitrite-BHA system was isolated and the structure was determined to be 2-tert-butyl-quinone. This compound gave a positive rec-assay test, and showed no mutagenesis by Ames' method. The active product from the nitrite-cysteine system was infered to be nitrosocysteine, and the product showed both DNA-damaging and mutagenic activity.