An accurate, rapid, and sensitive method for simultaneous determination of four typical heterocyclic amines in roasted pork patties: Application in the study of inhibitory effects of astaxanthin.

Heterocyclic aromatic amines, as a group of mutagenic and carcinogenic compounds, have gained worldwide concern. In this study, an accurate, rapid, and sensitive confirmation and quantification method of four major heterocyclic aromatic amines in roasted pork was developed based on Q-Orbitrap along with Quick, Easy, Cheap, Effective, Rugged, and Safe extraction. The limit of detections and limit of quantitations were found to be 0.2-1.2 µg/kg and 0.6-3.5 µg/kg, respectively, revealing the high sensitivity of this method. Obtained results showed recoveries ranging from 78.1 to 97.4%, depending on the different heterocyclic aromatic amines and spiked levels. Precision was in the range of 2.6-4.5% for four heterocyclic aromatic amines at different levels. In addition, the developed method had been applied to investigate the inhibitory effects of astaxanthin on the above-mentioned heterocyclic aromatic amines in roasted pork. The amount of astaxanthin with the best inhibitory effects was 7.5 mg (0.0375%), which led to significant reduction in heterocyclic aromatic amines levels over 50%.

CXCR4 inhibition in human pancreatic and colorectal cancers induces an integrated immune response.

Inhibition of the chemokine receptor CXCR4 in combination with blockade of the PD-1/PD-L1 T cell checkpoint induces T cell infiltration and anticancer responses in murine and human pancreatic cancer. Here we elucidate the mechanism by which CXCR4 inhibition affects the tumor immune microenvironment. In human immune cell-based chemotaxis assays, we find that CXCL12-stimulated CXCR4 inhibits the directed migration mediated by CXCR1, CXCR3, CXCR5, CXCR6, and CCR2, respectively, chemokine receptors expressed by all of the immune cell types that participate in an integrated immune response. Inhibiting CXCR4 in an experimental cancer medicine study by 1-wk continuous infusion of the small-molecule inhibitor AMD3100 (plerixafor) induces an integrated immune response that is detected by transcriptional analysis of paired biopsies of metastases from patients with microsatellite stable colorectal and pancreatic cancer. This integrated immune response occurs in three other examples of immune-mediated damage to noninfected tissues: Rejecting renal allografts, melanomas clinically responding to anti-PD1 antibody therapy, and microsatellite instable colorectal cancers. Thus, signaling by CXCR4 causes immune suppression in human pancreatic ductal adenocarcinoma and colorectal cancer by impairing the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells.

Defining the in vivo characteristics of acute myeloid leukemia cells behavior by intravital imaging.

The majority of acute myeloid leukemia (AML) patients have a poor response to conventional chemotherapy. The survival of chemoresistant cells is thought to depend on leukemia-bone marrow (BM) microenvironment interactions, which are not well understood. The CXCL12/CXCR4 axis has been proposed to support AML growth but was not studied at the single AML cell level. We recently showed that T-cell acute lymphoblastic leukemia (T-ALL) cells are highly motile in the BM; however, the characteristics of AML cell migration within the BM remain undefined. Here, we characterize the in vivo migratory behavior of AML cells and their response to chemotherapy and CXCR4 antagonism, using high-resolution 2-photon and confocal intravital microscopy of mouse calvarium BM and the well-established MLL-AF9-driven AML mouse model. We used the Notch1-driven T-ALL model as a benchmark comparison and AMD3100 for CXCR4 antagonism experiments. We show that AML cells are migratory, and in contrast with T-ALL, chemoresistant AML cells become less motile. Moreover, and in contrast with T-ALL, the in vivo exploratory behavior of expanding and chemoresistant AML cells is unaffected by AMD3100. These results expand our understanding of AML cells-BM microenvironment interactions, highlighting unique traits of leukemia of different lineages.

Fusion Stage of HIV-1 Entry Depends on Virus-Induced Cell Surface Exposure of Phosphatidylserine.

HIV-1 entry into host cells starts with interactions between the viral envelope glycoprotein (Env) and cellular CD4 receptors and coreceptors. Previous work has suggested that efficient HIV entry also depends on intracellular signaling, but this remains controversial. Here we report that formation of the pre-fusion Env-CD4-coreceptor complexes triggers non-apoptotic cell surface exposure of the membrane lipid phosphatidylserine (PS). HIV-1-induced PS redistribution depends on Ca2+ signaling triggered by Env-coreceptor interactions and involves the lipid scramblase TMEM16F. Externalized PS strongly promotes Env-mediated membrane fusion and HIV-1 infection. Blocking externalized PS or suppressing TMEM16F inhibited Env-mediated fusion. Exogenously added PS promoted fusion, with fusion dependence on PS being especially strong for cells with low surface density of coreceptors. These findings suggest that cell-surface PS acts as an important cofactor that promotes the fusogenic restructuring of pre-fusion complexes and likely focuses the infection on cells conducive to PS signaling.

Chemokine receptor CCR5 and CXCR4 might influence virus replication during IBDV infection.

Both CCR5 and CXCR4 are important chemokine receptors and take vital role in migration, development and distribution of T cells, however, whether they will influence the process of T cell infiltration into bursa of Fabricius during infectious bursal disease virus (IBDV) infection is unclear. In the current study, CCR5 and CXCR4 antagonists, Maraviroc and AMD3100, were administrated into chickens inoculated with IBDV, and the gene levels of IBDV VP2, CCR5, CXCR4 and related cytokines were determined by real-time PCR. The results showed that large number of T cells began to migrate into the bursae on Day 3 post infection with IBDV and the mRNA of chemokine receptors CCR5 and CXCR4 began to increase on Day 1. Moreover, antagonist treatments have increased the VP2, CCR5 and CXCR4 gene transcriptions and influenced on the gene levels of IL-2, IL-6, IL-8, IFN-γ, TGF-ß4, MHC-I and MDA5. In conclusion, the chemokine receptors CCR5 and CXCR4 might influence virus replication during IBDV infection and further study would focus on the interaction between chemokine receptors and their ligands.

Formation and mitigation of heterocyclic aromatic amines in fried pork.

Heterocyclic aromatic amines (HAAs) are potent mutagens and carcinogens generated during the heat processing of meat. HAAs, which are abundant in processed meat products, include 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP). The content of these three HAAs in fried pork was determined by LC-MS/MS. The effects of frying time and temperature, sample shape, and addition of antioxidants on the generation of HAAs were investigated. The results show that HAAs were produced during frying, and their levels increased with increasing frying time and temperature. Pork patties had the highest concentration of HAAs compared with pork meatballs and pork strips. The addition of antioxidant of bamboo leaves (AOB), liquorice extract, tea polyphenol, phytic acid and sodium iso-ascorbate to pork before frying had an inhibitory effect on HAA generation, with AOB being the most effective antioxidant. Inhibition levels of nearly 69.73% for MeIQx, 53.59% for 4,8-DiMeIQx and 77.07% for PhIP in fried pork were achieved when the concentrations of AOB added were 0.02, 0.01 and 0.10 g kg⁻¹, respectively.

Impact of environmental exposures on the mutagenicity/carcinogenicity of heterocyclic amines.

Carcinogenic heterocyclic amines are produced from overcooked foods and are highly mutagenic in most short-term test systems. One of the most abundant of these amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), induces breast, colon and prostate tumors in rats. Human dietary epidemiology studies suggest a strong correlation between either meat consumption or well-done muscle meat consumption and cancers of the colon, breast, stomach, lung and esophagus. For over 20 years our laboratory has helped define the human exposure to these dietary carcinogens. In this report we describe how various environmental exposures may modulate the risk from exposure to heterocyclic amines, especially PhIP. To assess the impact of foods on PhIP metabolism in humans, we developed an LC/MS/MS method to analyze the four major PhIP urinary metabolites following the consumption of a single portion of grilled chicken. Adding broccoli to the volunteers' diet altered the kinetics of PhIP metabolism. At the cellular level we have found that PhIP itself stimulates a significant estrogenic response in MCF-7 cells, but even more interestingly, co-incubation of the cells with herbal teas appear to enhance the response. Numerous environmental chemicals found in food or the atmosphere can impact the exposure, metabolism, and cell proliferation response of heterocyclic amines.

Inhibitory effects of beer on heterocyclic amine-induced mutagenesis and PhIP-induced aberrant crypt foci in rat colon.

Anti-mutagenic and anti-carcinogenic effects of beer on heterocyclic amine (HCA)-induced carcinogenesis were studied in vitro and in vivo. Four commercial beers (two pilsner-type, black, and stout) showed inhibitory effects against five HCAs, 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), in the Ames assay using Salmonella typhimurium TA98 in the presence of rat S9 mix. The inhibitory effects of dark-colored beers (stout and black beer) were greater than those of pilsner-type beers. Dark-colored beers suppressed CYP1A2 activity in a dose-dependent manner, suggesting that inhibition of HCA activation is partly responsible for their strong anti-mutagenic effects. Anti-mutagenic effects were also observed when the pooled human S9 mix or activated IQ was used in the assay. The micronucleus test using Chinese hamster lung CHL/IU cells showed that the addition of freeze-dried samples of pilsner-type and stout beer to the culture medium significantly reduced the number of cells with micronuclei induced with PhIP or Trp-P-2. Single-cell gel electrophoresis assay (comet assay) revealed that oral ingestion of pilsner-type and stout beers for 1 week significantly inhibited DNA damage in the liver cells of male ICR mice exposed to MeIQx (13 mg/kg, i.p.). A decrease in the formation of DNA adducts was also observed using a 32P-postlabeling method. Male Fischer 344 rats orally received PhIP (75 mg/kg, five times a week for 2 weeks) and aberrant crypt foci (ACF) formation in the colon was analyzed after 5 weeks. The number of ACF was significantly reduced in rats fed a diet containing freeze-dried beer. These results suggest that beer inhibits the genotoxic effects of HCAs and may reduce the risk of carcinogenesis caused by food borne carcinogens.

Development and application of test methods for the detection of dietary constituents which protect against heterocyclic aromatic amines.

This article describes the development and use of assay models in vitro (genotoxicity assay with genetically engineered cells and human hepatoma (HepG2) cells) and in vivo (genotoxicity and short-term carcinogenicity assays with rodents) for the identification of dietary constituents which protect against the genotoxic and carcinogenic effects of heterocyclic aromatic amines (HAs). The use of genetically engineered cells expressing enzymes responsible for the bioactivation of HAs enables the detection of dietary factors that inhibit the metabolic activation of HAs. Human derived hepatoma (HepG2) cells are sensitive towards HAs and express several enzymes [glutathione S-transferase (GST), N-acetyltransferase (NAT), sulfotransferase (SULT), UDP-glucuronosyltransferase (UDPGT), and cytochrome P450 isozymes] involved in the biotransformation of HAs. Hence these cells may reflect protective effects, which are due to inhibition of activating enzymes and/or induction of detoxifying enzymes. The SCGE assay with rodent cells has the advantage that HA-induced DNA damage can be monitored in a variety of organs which are targets for tumor induction by HAs. ACF and GST-P(+) foci constitute preneoplastic lesions that may develop into tumors. Therefore, agents that prevent the formation of these lesions may be anticarcinogens. The foci yield and the sensitivity of the system could be substantially increased by using a modified diet. The predictive value of the different in vitro and in vivo assays described here for the identification of HA-protective dietary substances relevant for humans is probably better than that of conventional in vitro test methods with enzyme homogenates. Nevertheless, the new test methods are not without shortcomings and these issues are critically discussed in the present article.

Inhibition of heterocyclic aromatic amine formation in fried ground beef patties by garlic and selected garlic-related sulfur compounds.

The effects of garlic and selected organosulfur compounds (diallyl disulfide, dipropyl disulfide, diallyl sulfide, allyl methyl sulfide, allyl mercaptan, cysteine, and cystine) on the formation of heterocyclic aromatic amines (HAAs) in fried ground beef patties were evaluated. Minced garlic cloves (ca. 4.8 to 16.7%, wt/wt) or organosulfur compounds (0.67 mmol) were added directly to ground beef. Patties (100 g) were fried at 225 degrees C (surface temperature) for 10 min per side. Two patties were fried for each replication, and five replicates were analyzed for each treatment. For each replicate, four subsamples were analyzed (two unspiked subsamples for concentration and two spiked subsamples for the recovery of HAA standards). The volatile sulfur compounds significantly (P < 0.05) reduced concentrations of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by reductions of 46 to 81%, while average reductions of 35, 22, and 71%, were achieved with cystine, cysteine, and whole garlic, respectively. The volatile sulfur compounds reduced concentrations of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by 34 to 67%, while reductions of 25, 19, and 63% (P < 0.05) were achieved with cystine, cysteine, and whole garlic, respectively. These studies confirm that garlic and some organosulfur compounds have the potential to reduce HAA formation incooked beef patties.

Antimutagenic activity of cacao: inhibitory effect of cacao liquor polyphenols on the mutagenic action of heterocyclic amines.

We investigated the effect of polyphenols derived from cacao liquor on the mutagenic action of heterocyclic amines (HCAs) in vitro and ex vivo. In the Ames test, the cacao liquor polyphenols showed antimutagenic effects in bacteria treated with HCA in the presence of an S-9 mixture; however, they showed less efficacy than quercetin. On the other hand, the cacao liquor polyphenols showed potent antimutagenic activity in bacteria treated with activated forms of HCA, compared with quercetin. We also evaluated the effect of these compounds on enzymatic activation of HCA. They weakly suppressed the production of activated HCA. In the host-mediated assay in mice, a method used to estimate the potential carcinogenicity of chemicals ex vivo, oral administration of the cacao liquor polyphenols, reduced the number of colonies of revertant bacteria recovered from the liver. These data suggest that the cacao liquor polyphenols have an antimutagenic effect not only in vitro, but also ex vivo.

N-Acetyltransferase genetics and their role in predisposition to aromatic and heterocyclic amine-induced carcinogenesis.

N-Acetyltransferases (EC 2.3.1.5) are important in both the activation and deactivation of aromatic and heterocyclic amine carcinogens. Two N-acetyltransferase isozymes (NAT1 and NAT2) encoded by NAT1 and NAT2, respectively, have been identified. Both NAT1 and NAT2 exhibit genetic polymorphisms, and recent investigations have increased our understanding of the relationship between genotype and phenotype. Several studies have shown a role for NAT1 and NAT2 acetylation polymorphisms in cancer risk in human populations, but the findings have been inconsistent. These findings may relate to variability in carcinogen exposures and to differences in acetylator genotype/phenotype determinations.

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.

Cytosolic activation of aromatic and heterocyclic amines. Inhibition by dicoumarol and enhancement in viral hepatitis B.

The aromatic amines 2-aminofluorene (2AF), 2-acetylaminofluorene, and 2-aminoanthracene, and the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, and 3-amino-1-methyl-SH-pyrido[4,3-b]indole (Trp-P-2) were activated by rat liver cytosolic fractions to form mutagenic metabolites in Salmonella typhimurium strains TA98, TA98NR, and TA98/1,8-DNP6. In the case of the Trp-P-2, the cytosolic activation was even more potent than the microsomal activation, which is classically ascribed to N-hydroxylation and subsequent esterification. The cytosolic activation was a) NADPH-dependent, b) induced by pretreatment of rats with 3-methylcholanthrene and especially Aroclor 1254 but not by phenobarbital, and c) inhibited by dicoumarol. The hypothesis is that, following a preliminary oxidative step in the cytosol (pure cytosolic activation) or in microsomes via prostaglandin H synthase (mixed microsomal-cytosolic activation), an oxidized intermediate of amino compounds may serve as substrate for DT diaphorase activity and bielectronically reduced to the corresponding N-hydroxyamino derivative. Purified DT diaphorase, in the presence of either NADPH or NADH as electron donor, produced mutagenic derivatives from IQ and Trp-P-2. An NADPH-dependent activation of Trp-P-2 also occurred in the liver cytosol of woodchucks (Marmota monax), but was not inhibited by dicoumarol. As previously demonstrated with liver S-12 fractions in both humans and woodchucks, the cytosolic activation of Trp-P-2 was enhanced in animals affected by hepatitis B virus infection. This enhanced metabolism, which persisted even after appearance of primary hepatocellular carcinoma in virus carriers, is likely to be ascribed to mechanisms other than DT diaphorase induction, such as glutathione depletion.

Inhibitory activity of cigarette-smoke condensate on the mutagenicity of heterocyclic amines.

Cigarette-smoke condensate (CSC) is a complex mixture containing over 3800 identified chemicals including nicotine, water, mutagens, antimutagens, cytotoxins and inert chemicals. Although CSC is mutagenic in the Ames test, its effect on the activity of other mutagens has not been characterized. Using the Ames Salmonella bacterial mutagenesis assay, we found CSC exerts a significant inhibitory effect on mutagens requiring bioactivation. Those studied included heterocyclic amines (Glu-P-1, Glu-P-2, IQ, MeIQ, Trp-P-1 and Trp-P-2), benzo[a]pyrene (B[a]P) and aflatoxin B1. However, CSC had no effect on the activity of direct-acting mutagens (2-nitrofluorene, sodium azide, 4-nitro-1,2-phenylenediamine, 4-nitroquinoline N-oxide and methyl methanesulfonate). With indirect-acting mutagens, the reduced number of revertants observed in the presence of CSC was not attributable to cytotoxicity. CSC exhibited a potent inhibitory effect on the cytochrome P-450 dependent monooxygenases, ethoxyresorufin O-deethylase and B[a]P hydroxylase. This suggests inhibition of the cytochrome P-450 isozymes as one possible mechanism for the antimutagenicity of CSC. Fractionation studies of CSC revealed that the neutral, weakly acidic (phenolic) and basic fractions are all effective as antimutagens against Glu-P-1, a representative heterocyclic amine. This indicates that several classes of chemicals contribute to the inhibitory effect of CSC on the mutagenicity of the heterocyclic amines.

Substances in human urine that strongly inhibit bacterial mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and related heterocyclic amines.

Extracts of human urine were shown to contain substances that strongly inhibited the liver S9-mediated mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in Salmonella typhimurium TA98 strain in a liquid incubation assay. The inhibitory effect was unrelated to cytotoxicity and was similar with urine extracts from smokers and non-smokers. Under similar assay conditions, the mutagenicity of the related amino-imidazoazaarenes, 2-amino-3-methyl-imidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline was also found to be strongly inhibited by urine extracts. Decreased or enhanced mutagenicity was seen with 2-acetyl-aminofluorene and 2-aminoanthracene depending on the type of assay, and the time of incubation in liquid medium. A weak inhibition of the mutagenicity of 2-nitrofluorene, a direct-acting mutagen, was observed only after a short incubation time. Mutagenicity of 4-nitroquinoline N-oxide was not altered by the presence of urine extracts at concentrations shown to be inhibitory for the mutagenicity of heterocyclic aromatic amines. Our data suggest that the inhibitory substances in urine act through their capacity to non covalently bind the parent heterocyclic and aromatic amines, thus affecting their availability in aqueous medium for diffusion into liver microsomes where metabolic activation takes place.

Nickel-mediated inhibition in the glutathione-dependent protection against lipid peroxidation.

Glutathione protects liver microsomes against the rapid onset of lipid peroxidation via a sulfhydryl dependent heat labile factor known as free radical reductase. The administration of nickel to mice resulted in an inhibition in the activity of free radical reductase, and enhanced lipid peroxidation and the activity of glutathione S-transferase in a dose dependent manner. The pretreatment of cyclam, a known specific chelator of nickel restored free radical reductase and glutathione S-transferase activities and alleviated nickel mediated enhancement of lipid peroxidation. Our results indicate that nickel-mediated inhibition in free radical reductase activity and activation of glutathione S-transferase may be due to the interaction of nickel with sensitive-SH groups located on these proteins.

Use of nitrite and hypochlorite treatments in determination of the contributions of IQ-type and non-IQ-type heterocyclic amines to the mutagenicities in crude pyrolyzed materials.

The mutagenic heterocyclic amines Glu-P-2, MeA alpha C and Phe-P-1, which possess a 2-aminopyridine structure in their molecule (non-IQ-type mutagens), were found to be inactivated by nitrite treatment under acidic conditions, as observed previously with Trp-P-1, Trp-P-2, Glu-P-1 and A alpha C. In contrast, MeIQx, 4,8- and 7,8-DiMeIQx, which were originally isolated from fried beef or heated model mixtures of creatinine, amino acids and glucose, and which have a 2-aminoimidazole moiety in their molecules (IQ-type mutagens), were very resistant to nitrite treatment like IQ and MeIQ. Both types of mutagenic heterocyclic amines were completely inactivated by treatment with hypochlorite. This differential inactivation of mutagenic heterocyclic amines by nitrite and hypochlorite was used in determination of the contributions of IQ-type and non-IQ-type mutagens to the total mutagenicities of various pyrolyzed materials. The percentage contributions of IQ-type mutagens to the mutagenicities of broiled sardine, fried beef, broiled horse mackerel, cigarette smoke condensate and albumin tar were 88, 75, 48, 6 and 4, respectively.