Evoking cytochrome P450 1A activity interferes with apoptosis induced by 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp-P-1) in rat hepatocytes under the ex vivo system

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is known as a dietary carcinogen and it requires metabolic activation by cytochrome P450 (CYP) 1A subfamily to have carcinogenicity. On the other hand, our previous report demonstrated that Trp-P-1 induces apoptosis in primary cultured rat hepatocytes, but the metabolically activated Trp-P-1 added extracelluarly to hepatocytes did not induce apoptosis. In this study, we focused on the intracellular status of CYPs and investigated apoptotic events induced by Trp-P-1 using hepatocytes isolated from rats treated with three chemical inducers for CYPs. In cultured hepatocytes from rats treated with 3-methylchoranthrene, which mainly induces CYP 1A, Trp-P-1-induced apoptosis was suppressed. In the same cultures, intact Trp-P-1 was decreased and its metabolites were increased. Phenobarbital and pyridine did not affect Trp-P-1-induced apoptosis. These results suggested that evoking CYP 1A activity might interfere with apoptosis induced by Trp-P-1 in rat hepatocytes under the ex vivo system.     

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 LD₅₀ at 6 h in culture was 15 μM, and the time for the 50% decrease in cell viability (t_1/2) at 20 μM was 3 h. 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 occured in splenocytes, but not in thymocytes although Trp-P-1 activated 32–34 kDa 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.     

3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (trp-P-1) is incorporated into rat splenocytes,thymocytes, and hepatocytes through monoamine transporters and induces apoptosis

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which is a tryptophan pyrolysate formed during cooking, induces apoptosis in rat splenocytes, thymocytes, and hepatocytes. In this study, we investigated whether Trp-P-1 is transported into these cells and causes apoptosis. Trp-P-1 was immediately incorporated into rat splenocytes, thymocytes, and hepatocytes in a dose- and time-dependent manner. Dopamine and serotonin significantly competed with the uptake of Trp-P-1 into these cells, and nomifensine and indatraline, which are inhibitors of dopamine- and serotonin-transporters, respectively, markedly suppressed the uptake of Trp-P-1. On the other hand, amino acids including tryptophan did not compete with Trp-P-1. Inhibition of monoamine transporters using nomifensine and indatraline partially suppressed Trp-P-1-induced cell death in these cells. In hepatocytes, the inhibition of transporters prevented Trp-P-1-induced morphological changes and activation of caspase-3. These results demonstrated that Trp-P-1 is incorporated into the cells through monoamine transporters and induces apoptosis.     

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 LD₅₀ at 6 h in culture was 15 μM, and the time for the 50% decrease in cell viability (t_1/2) at 20 μM was 3 h. 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 occured in splenocytes, but not in thymocytes although Trp-P-1 activated 32–34 kDa 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.     

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 induces apoptosis and necrosis with activation of different caspases in rat splenocytes

A dietary carcinogen, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) at 20 microM activates caspase-3-like proteases as an apoptotic marker in rat splenocytes. The present study demonstrated 100 microM Trp-P-1 induced necrosis with activation of caspase-3-like proteases. The activation in necrosis and apoptosis resulted from the activation of caspase-9 and caspase-8, respectively. Thus, Trp-P-1 induces apoptosis and necrosis with the activation of different caspases.     

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole Induces Apoptosis and Necrosis with Activation of Different Caspases in Rat Splenocytes

A dietary carcinogen, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) at 20 μM activates caspase-3-like proteases as an apoptotic marker in rat splenocytes. The present study demonstrated 100 μM Trp-P-1 induced necrosis with activation of caspase-3-like proteases. The activation in necrosis and apoptosis resulted from the activation of caspase-9 and caspase-8, respectively. Thus, Trp-P-1 induces apoptosis and necrosis with the activation of different caspases.     

Interspecies homology of cytochrome P-450: toxicological significance of cytochrome P-450 cross-reactive with anti-rat P-448-H antibodies in liver microsomes from dogs, monkeys and humans

The mutagenic activation of various promutagens by liver microsomes from dogs, monkeys and humans was investigated. Dog liver microsomes efficiently catalyzed the mutagenic activation of Trp-P-2 and Glu-P-1 followed by IQ and AAF. Monkey liver microsomes were most active in the activation of IQ followed by Glu-P-1, AAF and Trp-P-2. Although there were remarkable individual differences, human liver microsomes were found to be most active in the mutagenic activation of IQ followed by Trp-P-2, Glu-P-1 and AAF. Antibodies against rat P-448-H inhibited the mutagenic activation of Glu-P-1, Trp-P-2 and IQ in rat and dog liver microsomes, and Glu-P-1 and Trp-P-2 in monkey liver microsomes. The activation of Glu-P-1 and IQ in human liver microsomes was also strongly inhibited by anti-P-448-H antibodies. The amounts of cytochrome P-450 cross-reactive with anti-P-448-H antibodies in human liver microsomes highly correlated with the capacity to activate Glu-P-1, Trp-P-2 and IQ but not AAF.     

Antimutagenicity of sweetpotato (Ipomoea batatas) roots

Antimutagenicity of the water extracts prepared from the storage roots of four varieties of sweetpotato with different flesh colors was investigated using Salmonella typhimurium TA 98. The extract from the whole roots of the purple-colored Ayamurasaki variety effectively decreased the reverse mutation induced not only by Trp-P-1, Trp-P-2, IQ, B[a]P, and 4-NQO but also by dimethyl sulfoxide extracts of grilled beef. Comparison of the inhibitory activity of the extracts from the normal Ayamurasaki and its anthocyanin-deficient mutant one suggested that the anthocyanin pigment in the flesh decreases the mutagenic activity of the mutagens as heterocyclic amines. Two anthocyanin pigments purified from purple-colored sweet-potato, 3-(6,6'-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and peonidin (YGM-6) effectively inhibited the reverse mutation induced by heterocyclic amines, Trp-P-1, Trp-P-2, and IQ in the presence of rat liver microsomal activation systems.     

Mutagenic activation of 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole(Trp-P-1) and 3-amino-1-methyl-5H-pyrido(4,3-b)indole (Trp-P-2) by primary cultures of adult rat hepatocytes: effect of Aroclor induction in vitro

The mutagenic activation of tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, was studied in a Salmonella TA98/hepatocyte mutagenesis assay. Adult rat hepatocytes in primary culture were either untreated or induced by the addition of Aroclor 1254 (2 micrograms/ml) 18-20 h before the mutagenesis test which was performed at day 1 and at day 2 after the isolation of hepatocytes. The mutagenic activation of Trp-P-1 and Trp-P-2 was studied as a function of the time of incubation and of the concentration of chemical. Trp-P-1 and Trp-P-2 incubated for 20 min in the presence of untreated hepatocytes and bacteria gave rise to a weak number of revertants which doubled the level of spontaneous mutants. Aroclor-induced hepatocytes became highly competent in mutagenic activation of tryptophan pyrolysis products and the induction ratio reached 4.9 and 7.1 for Trp-P-1 and Trp-P-2, respectively, after 60 min of incubation, on day 2 of the experiment. It should be noted that the induction ratio was higher on day 2 than on day 1. When conditions were standardized, i.e. Aroclor-induced hepatocytes on day 2, final concentration of cellular protein about 1 mg/ml, 20 min of incubation, the Salmonella/hepatocyte assay produced a linear concentration-dependent mutagenic response for Trp-P-1 and Trp-P-2. By comparing the results obtained with Aroclor-induced hepatocytes and Aroclor-induced liver S9 fraction in the Salmonella test, it could be estimated that hepatocytes were 3 times less active than the S9 fraction with regard to mutagenic activation of both Trp-P-1 and Trp-P-2.     

In vivo genotoxicity of heterocyclic amines detected by a modified alkaline single cell gel electrophoresis assay in a multiple organ study in the mouse

We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of 6 heterocyclic amines, Trp-P-1 (25 mg/kg), Trp-P-2 (13 mg/kg), IQ (13 mg/kg), MeIQ (13 mg/kg), MeIQx (13 mg/kg) and PhIP (40 mg/kg), in mouse liver, lung, kidney, brain, spleen, bone marrow and stomach mucosa. Mice were sacrificed 1, 3, and 24 h after intraperitoneal injection. Trp-P-2, IQ, MeIQ, and MeIQx yielded statistically significant DNA damage in the stomach, liver, kidney, lung and brain; Trp-P-1 in the stomach, liver and lung; and PhIP in the liver, kidney and brain. None of the heterocyclic amines induced DNA damage in the spleen and bone marrow. Our results suggest that the alkaline SCG assay applied to multiple organs is a good way to detect organ-specific genotoxicity of heterocyclic amines in mammals.     

The Piperaceae Amides I: Structure of Pipercide,A New Insecticidal Amide from Piper nigrum L.

It was evidenced that mutagenic principles in tryptophan pyrolysate, 3-amino-1,4-dimethyl-5H pyrido(4,3-b) indole and 3-amino-1-methyl-5H pyrido(4,3-b) indole (abbreviated as Trp-P-1 and Trp-P-2, respectively) bind to DNA without activation by rat liver microsomes. The bindings of Trp-P-1 and Trp-P-2 were not random and did not introduce strand scissions into DNA. Trp-P-1 bound more easily than Trp-P-2. The bindings of these mutagenic principles to DNA were concluded by using negatively superhelical simian virus 40 (SV40) DNA from following experimental data. (1) The intensity of ethidium bromide (EtBr)-DNA fluorescence by illumination with UV light and the electrophoretic mobility of superhelical DNA in agarose gel decreased as a function of the amounts of Trp-P-1 and Trp-P-2. (2) In vitro RNA synthesis catalyzed by Escherichia coli DNA-dependent RNA polymerase and nick-translation catalyzed by Escherichia coli DNA polymerase I (Kornberg enzyme) were inhibited significantly on DNA treated with Trp-P-1 and Trp-P-2. (3) The negative superhelicity of SV40 DNA introduces unpaired regions into DNA. These regions can be cleaved by single-strand-specific S1 endonuclease to generate unit length linear duplex molecules. It was found that this S1-sensitivity of DNA decreased by treatment with Trp-P-1. (4) The cleavage patterns of Trp-P-1 treated DNA with five restriction endonucleases were investigated. The protection of the cleavage site by the drug was observed against HincII, HindIII and EcoRII, whereas not against HaeIII and HinfI. These results show that the binding of Trp-P-1 to DNA is not random. Identical results were also obtained in Trp-P-2.

However, the bindings of Trp-P-1 and Trp-P-2 were not so tight, and phenol extraction of the complex dissociated these drugs from DNA.     

Activation of a mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole. Identification of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole and its reaction with DNA

A potent mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), isolated from a tryptophan pyrolysate, was activated metabolically by rat liver microsomes and bound to DNA. An active metabolite formed by rat liver microsomes was identified as 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2). Synthetic N-OH-Trp-P-2 reacted with DNA efficiently after O-acetylation or to a lesser extent under acidic conditions (pH 5.5), but did not react appreciably under neutral conditions. Acid hydrolysis of DNA modified by O-acetylated N-OH-Trp-P-2 (N-OAc-Trp-P-2) gave 3-(8-guanyl)amino-1-methyl-5H-pyrido[4,3-b]indole (Gua-Trp-P-2), which is the main modified base of DNA formed by Trp-P-2 in the presence of microsomes. The glycoside bond of the modified base was found to be cleaved by heating at 100° for 1 hr at pH 7.0. In this way, the modified base was liberated from DNA modified by N-OAc-Trp-P-2 in good yield. N-OAc-Trp-P-2 bound to guanyl cytidine more effectively than to guanylic acid, suggesting that covalent binding with guanyl moiety of DNA involves intercalation of the ultimate mutagen into a base pair.     

Isolation and characterization of active metabolites of tryptophan-pyrolysate mutagen,TRP-P-2, formed by rat liver microsomes

The mutagenic compound derived from the pyrolysis of tryptophan, 3-amino-1-methyl-5H-pyrido-[4,3b]indole (Trp-P-2) was metabolized by rat liver microsomes to more than four metabolites, separable by high performance liquid chromatography. Among these metabolites, two metabolites, M-3 and M-4 were directly active in increasing the frequency of mutation in Salmonella typhimurium TA98. Treatments of rats with polychlorinated biphenyl (PCB) or 3-methylcholanthrene dramatically induced the activity of liver microsomes to form these active metabolites, while treatment with phenobarbital was without effect. A major active metabolite (M-3) formed the pentacyano-ammine ferroate, which is known to be formed by reaction of sodium pentacyano-ammine ferroate with some hydroxylamines. Further this metabolite was oxidized to the minor active metabolite (M-4) with potassium ferricyanide or γ-manganese dioxide, and was reduced back to Trp-P-2 with titanium trichloride. These results indicated that the major active metabolite of Trp-P-2, which is formed by cytochrome P-450, is the 3-hydroxyamino derivative.     

Metabolic activation of 3-amino-5H-pyrido[4,3-b]indole, a highly mutagenic principle in tryptophan pyrolysate, by rat liver enzymes.

3-Amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), a mutagenic principle in tryptophan pyrolysates, binds to DNA after metabolic activation by rat liver enzymes. The enzymes for activation of Trp-P-2 were found in both microsomes and the cytosol. The reaction required NADPH and ATP, metabolic and was inhibited by 7,8-benzoflavone. Considerable binding was observed with only microsomes as enzyme source, but further addition of cytosol enhanced the binding, enhancement depending on the amount of cytosol added. Inducers for microsomal mixed-function oxidases induced activating enzyme(s) for Trp-P-2, 3-methylcholanthrene being most effective, followed by polychlorinated biphenyls and then phenobarbital.     

Inhibition of mutagenicity of food-derived heterocyclic amines by sulforaphane--a constituent of broccoli

Sulforaphane, a constituent of broccoli was investigated for its antimutagenic potential against different classes of cooked food mutagens (heterocyclic amines). These include imidazoazaarenes such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); pyridoindole derivatives such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2); and, dipyridoimidazole derivative such as 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1). Tests were carried out by Ames Salmonella/reversion assay using Salmonella typhimurium TA98 (frame shift mutation sensitive) and TA100 (base pair mutation sensitive) bacterial strains in the presence of Aroclor 1254-induced rat liver S9. Results of these in vitro antimutagenicity studies strongly suggest that sulforaphane is a potent inhibitor of the mutagenicity induced by imidazoazaarenes such as IQ, MeIQ and MeIQx (approximately 60% inhibition) and moderately active against pyridoindole derivatives such as Trp-P-1 and Trp-P-2 (32-48% inhibition), but ineffective against dipyridoimidazole derivative (Glu-P-1) in TA 100.     

Reduction of enzyme activity of tyrosine hydroxylase and aromatic L-aminoacid decarboxylase in clonal pheochromocytoma PC12h cells by carcinogenic heterocyclic amines

Out of carcinogenic heterocyclic amines, which are produced by pyrolysis of tryptophan in food, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) were found to reduce the activity of enzymes related to catecholamine metabolism in clonal rat pheochromocytoma PC12h cells. By 6 days' culture in the presence of 10 nM to 10 microM Typ-P-1 and -2, these heterocyclic amines were accumulated in the cells, and activity of tyrosine hydroxylase (TH) and aromatic L-aminoacid decarboxylase (AADC) were reduced markedly. Reduction of these enzyme activity was observed with Trp-P-1 and -2 at the concentrations lower than 1 microM, while cell protein and enzyme activity of a non-specific enzyme, beta-galactosidase were reduced only with 10 microM Trp-P-1. These results show that these heterocyclic amines are neurotoxins specific for dopaminergic neurons.     

Effect of glutathione and uridine-5′-diphosphoglucuronic acid on the mutagenicity of tryptophan pyrolysis products (Trp-P-1 and Trp-P-2) by rat-liver and -intestine S9 fraction

In the Ames test, after the addition of glutathione (GSH) or uridine-5′ diphosphoglucuronic acid (UD-PGA), we observed for Trp-P-1 an unchanged or a reduced mutagenicity by both the liver and intestine S9 fraction. For Trp-P-2, the same was true when we used the intestine S9 fraction. In the presence of liver S9 fraction, Trp-P-2 mutagenicity was also decreased by the addition of UDPGA but was increased by the addition of GSH. These results show that cofactors for glucuronide and GSH conjugation may alter the metabolic activation of Trp-P-1 and Trp-P-2 and consequently their mutagenicity.     

Effect of glutathione and uridine-5'-diphosphoglucuronic acid on the mutagenicity of tryptophan pyrolysis products (Trp-P-1 and Trp-P-2) by rat-liver and -intestine S9 fraction

In the Ames test, after the addition of glutathione (GSH) or uridine-5'-diphosphoglucuronic acid (UD-PGA), we observed for Trp-P-1 an unchanged or a reduced mutagenicity by both the liver and intestine S9 fraction. For Trp-P-2, the same was true when we used the intestine S9 fraction. In the presence of liver S9 fraction, Trp-P-2 mutagenicity was also decreased by the addition of UDPGA but was increased by the addition of GSH. These results show that cofactors for glucuronide and GSH conjugation may alter the metabolic activation of Trp-P-1 and Trp-P-2 and consequently their mutagenicity.     

Developmental changes in hepatic activation of dietary mutagens by mice

Metabolic activation of the food mutagens 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and aflatoxin B1 by female BALB/c mice of different ages (2-24 weeks) was investigated in vivo and in vitro using Salmonella typhimurium TA98 as the indicator organism. The in vivo activation of the three mutagens was investigated in 4- and 24-week-old mice using an intrasanguineous host-mediated assay. All three compounds showed reduced levels of activation with the older hosts. Hepatic S9 fractions from female mice of varying ages between 2 and 24 weeks were used in the in vitro mutagenicity assay. To achieve optimal activation to bacterial mutagens, 5% S9 was required for aflatoxin B1 and Trp-P-2 and 10% S9 for MeIQ; age of donor generally had little effect on the profile of these protein activation curves. Under these optimal conditions MeIQ and Trp-P-2 both exhibited, as before, age-dependent decreases in activation over a wide range of mutagen concentrations, however the in vitro activation of aflatoxin showed no consistent change with age. Spectrophotometric measurements of S9 cytochrome P-450 content showed a decrease in concentration with increasing age, but this was not sufficient to account for changes observed in hepatic mutagen activation. However, changes in the activities of certain cytochrome P-450 isoenzymes and cytosolic GSH-transferases, which in turn result in changes in the activation and detoxification capacity of the liver, would appear to explain age-dependent changes in the activity of mutagens in vivo.