Isolation of two new stereochemical variants of streptophenazine by cocultivation of Streptomyces NIIST-D31, Streptomyces NIIST-D47, and Streptomyces NIIST-D63 strains in 3C2 combinations.

Cocultivation of combinations of Streptomyces species isolated from the same soil was explored to isolate novel secondary metabolites. Recently, we reported the isolation of a novel vicinal diepoxide of alloaureothin along with three carboxamides, 4-aminobenzoic acid, and 1,6-dimethoxyphenazine from the individual culture of Streptomyces luteireticuli NIIST-D31. Herein, cocultivation of NIIST-D31 with Streptomyces luteoverticillatus NIIST-D47 afforded two new stereochemical variants of streptophenazine (S1 and S2), and 1-N-methylalbonoursin, where the individual culture of NIIST-D47 primarily produced carbazomycins A, D, and E. The new streptophenazines and 1-N-methylalbonoursin were also observed during cocultivation of NIIST-D31 with Streptomyces thioluteus NIIST-D63, where the individual culture of NIIST-D63 strain afforded for the first time 2,2'-bipyridines (caerulomycinamide and dipyrimicin B), picolinamide, 2,3-dimethoxybenzamide, 2-hydroxy-3-methoxybenzamide, and 6-amino-2-pyridone along with known natural products aureothin and 1,6-dimethoxyphenazine. Finally, cocultivation of NIIST-D47 and NIIST-D63 strains produced carbazomycins B and C, alloaureothin, cyclo-(Leu-Pro), investiamide, and 4-aminobenzoic acid. Some of the compounds observed in the individual cultures were also produced in cocultivations. Improvement in the yield of secondary metabolites during cocultivation compared to individual culturing is well-known, which is noted here for vicinal diepoxide of alloaureothin. The production of new streptophenazines by cocultivation combinations with NIIST-D31 suggests that NIIST-D47 and NIIST-D63 may function as inducers in activating cryptic secondary metabolite-biosynthetic gene clusters. Cytotoxicity of the new streptophenazines in cancerous (MCF7 and MDA-MB-231) or non-cancerous (WI-38) cells were tested, however, they exhibited no significant activity.

Expression of arylamine N-acetyltransferase 2 activity in immortalized human bronchial epithelial cells.

Lung cancer is the leading cause of cancer deaths in the United States with high incidence in tobacco smokers. Arylamine N-acetyltransferase 2 (NAT2) is a xenobiotic enzyme that catalyzes both N- and O-acetylation of carcinogens present in tobacco smoke and contributes towards the genotoxicity of these carcinogens. NAT2 allelic variants result in slow, intermediate, and rapid acetylation phenotypes. A recent meta-analysis reported NAT2 non-rapid (slow and intermediate) phenotypes had a significantly increased risk of lung cancer. NAT2 activity in humans is thought to be restricted to liver and gastrointestinal tract, and no studies to our knowledge have reported the expression of NAT2 activity in immortalized human lung epithelial cells. Given the importance of NAT2 in cancer and inhalation of various carcinogens directly into the lungs, we investigated NAT2 activity in human lung epithelial cells. Both NAT1 and NAT2 protein were detected by "in-cell" Western. Arylamine N-acetyltransferase activity was determined with selective substrates for NAT1 (p-aminobenzoic acid; PABA) and NAT2 (sulfamethazine; SMZ) in the presence and absence of a selective NAT1 inhibitor. PABA N-acetylation (NAT1 activity) in cell protein lysates was abolished in the presence of 25 µM of NAT1 inhibitor whereas SMZ N-acetylation (NAT2) was unaffected. Incubation with the NAT1 inhibitor partially reduced the N-acetylation of ß-naphthylamine and the O-acetylation of N-hydroxy-4-aminobiphenyl consistent with catalysis by both NAT1 and NAT2. Immortalized human lung epithelial cells exhibited dose-dependent N-acetylation of 4-ABP with an apparent KM of 24.4 ± 5.1 µM. These data establish that NAT2 is expressed and functional in immortalized human lung epithelial cells and will help us further our understanding of NAT2 in lung cancer.

Construction and in vitro characterisation of aroA defective (aroAΔ) mutant Salmonella Infantis.

Poultry vaccine programs are important for control of Salmonella infections. Although there are vaccines for Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Typhi, there are no vaccines for Salmonella Infantis which has an increased rate in the world. In this study, it was aimed to generate aroA gene deleted mutant bacteria for the constitution of S. Infantis vaccine prototype and the in vitro characterisation of this bacterium. S. Infantis auxotrophic mutant which has a block at any step of chorismate pathway has been constituted for the first time in the world and it was determined that this bacterium gets susceptibility against some antibiotics and antimicrobial substances. It was also observed that the adhesion and invasion rate of mutant strain tenfold decreased in comparison with the field strain in cell culture assay. It is understood from the in vitro evaluation of this mutant strain that it can be used as a vaccine candidate in further vaccine development studies.

Root Gravitropism Is Regulated by a Crosstalk between para-Aminobenzoic Acid, Ethylene, and Auxin.

Plants respond to gravitational force through directional growth along the gravity vector. Although auxin is the central component of the root graviresponse, it works in concert with other plant hormones. Here, we show that the folate precursor para-aminobenzoic acid (PABA) is a key modulator of the auxin-ethylene interplay during root gravitropism in Arabidopsis (Arabidopsis thaliana). In gravistimulated roots, PABA promotes an asymmetric auxin response, which causes the asymmetric growth responsible for root curvature. This activity requires the auxin response transcription factors AUXIN RESPONSE FACTOR7 (ARF7) and ARF19 as well as ethylene biosynthesis and signaling, indicating that PABA activity requires both auxin and ethylene pathways. Similar to ethylene, exogenous PABA reverses the agravitropic root growth of the auxin transport mutant pin-formed2 (pin2) and the auxin biosynthetic double mutant with loss of function of weak ethylene insensitive (wei) genes, wei8wei2, but not the pin2wei8wei2 triple mutant. This finding suggests that PABA regulates the ethylene-dependent reciprocal compensation between auxin transport and biosynthesis. Furthermore, manipulation of endogenous free PABA levels by modulating the expression of the gene encoding its glucosylation enzyme, UDP-GLYCOSYL TRANSFERASE75B1, impacts the root graviresponse, suggesting that endogenous free PABA levels may play a crucial role in modulating the auxin-ethylene cross talk necessary for root gravitropism.

Arylamine N-acetyltransferase 1 in situ N-acetylation on CD3+ peripheral blood mononuclear cells correlate with NATb mRNA and NAT1 haplotype.

Human arylamine N-acetyltransferase 1 (NAT1) is responsible for the activation and elimination of xenobiotic compounds and carcinogens. Genetic polymorphisms in NAT1 modify both drug efficacy and toxicity. Previous studies have suggested a role for NAT1 in the development of several diseases. The aim of the present study was to evaluate NAT1 protein expression and in situ N-acetylation capacity in peripheral blood mononuclear cells (PBMC), as well as their possible associations with the expression of NAT1 transcript and NAT1 genotype. We report NAT1 protein, mRNA levels, and N-acetylation in situ activity for PBMC obtained from healthy donors. NAT1-specific protein expression was higher in CD3+ cells than other major immune cell subtypes (CD19 or CD56 cells). N-acetylation of pABA varied markedly among the PBMC of participants, but correlated very significantly with levels of NAT1 transcripts. NAT1*4 subjects showed significantly (p = 0.017) higher apparent pABA V max of 71.3 ± 3.7 versus the NAT1*14B subjects apparent V max of 58.5 ± 2.5 nmoles Ac-pABA/24 h/million cells. Levels of pABA N-acetylation activity at each concentration of substrate evaluated also significantly correlated with NAT1 mRNA levels for all samples (p < 0.0001). This highly significant correlation was maintained for samples with the NAT1*4 (p = 0.002) and NAT1*14B haplotypes (p = 0.0106). These results provide the first documentation that NAT1-catalyzed N-acetylation in PBMC is higher in T cell than in other immune cell subtypes and that individual variation in N-acetylation capacity is dependent upon NAT1 mRNA and NAT1 haplotype.

Folate Acts in E. coli to Accelerate C. elegans Aging Independently of Bacterial Biosynthesis.

Folates are cofactors for biosynthetic enzymes in all eukaryotic and prokaryotic cells. Animals cannot synthesize folate and must acquire it from their diet or microbiota. Previously, we showed that inhibiting E. coli folate synthesis increases C. elegans lifespan. Here, we show that restriction or supplementation of C. elegans folate does not influence lifespan. Thus, folate is required in E. coli to shorten worm lifespan. Bacterial proliferation in the intestine has been proposed as a mechanism for the life-shortening influence of E. coli. However, we found no correlation between C. elegans survival and bacterial growth in a screen of 1,000+ E. coli deletion mutants. Nine mutants increased worm lifespan robustly, suggesting specific gene regulation is required for the life-shortening activity of E. coli. Disrupting the biosynthetic folate cycle did not increase lifespan. Thus, folate acts through a growth-independent route in E. coli to accelerate animal aging.

PABA/NO lead optimization: Improved targeting of cytotoxicity to glutathione S-transferase P1-overexpressing cancer cells.

PABA/NO [O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino) diazen-1-ium-1,2-diolate] is a nitric oxide (NO)-releasing arylating agent designed to be selectively activated by reaction with glutathione (GSH) on catalysis by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancer cells. PABA/NO has proven active in several cancer models in vitro and in vivo, but its tendency to be metabolized via a variety of pathways, some that generate inactive metabolites and hydrolysis products, limits its potential as a drug. Here we show that a simple replacement of cyano for nitro at the 4 position to give compound 4b ('p-cyano-PABA/NO') has the dual effect of slowing the undesired side reactions while enhancing the proportion of NO release and arylating activity on catalysis by GSTP1. Compound 4b showed increased resistance to hydrolysis and uncatalyzed reaction with GSH, along with a more favorable product distribution in the presence of GSTP1. It also showed significant proapoptotic activity. The data suggest p-cyano-PABA/NO to be a more promising prodrug than PABA/NO, with better selectivity toward cancer cells.

Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.

Coq9 is a polypeptide subunit in a mitochondrial multi-subunit complex, termed the CoQ-synthome, required for biosynthesis of coenzyme Q (ubiquinone or Q). Deletion of COQ9 results in dissociation of the CoQ-synthome, but over-expression of Coq8 putative kinase stabilizes the CoQ-synthome in the coq9 null mutant and leads to the accumulation of two nitrogen-containing Q intermediates, imino-demethoxy-Q6 (IDMQ6) and 3-hexaprenyl-4-aminophenol (4-AP) when para-aminobenzoic acid (pABA) is provided as a ring precursor. To investigate whether Coq9 is responsible for deamination steps in Q biosynthesis, we utilized the yeast coq5-5 point mutant. The yeast coq5-5 point mutant is defective in the C-methyltransferase step of Q biosynthesis but retains normal steady-state levels of the Coq5 polypeptide. Here, we show that when high amounts of 13C6-pABA are provided, the coq5-5 mutant accumulates both 13C6-imino-demethyl-demethoxy-Q6 (13C6-IDDMQ6) and 13C6-demethyl-demethoxy-Q6 (13C6-DDMQ6). Deletion of COQ9 in the yeast coq5-5 mutant along with Coq8 over-expression and 13C6- pABA labeling leads to the absence of 13C6-DDMQ6, and the nitrogen-containing intermediates 13C6-4-AP and 13C6-IDDMQ6 persist. We describe a coq9 temperature-sensitive mutant and show that at the non-permissive temperature, steady-state polypeptide levels of Coq9-ts19 increased, while Coq4, Coq5, Coq6, and Coq7 decreased. The coq9-ts19 mutant had decreased Q6 content and increased levels of nitrogen-containing intermediates. These findings identify Coq9 as a multi-functional protein that is required for the function of Coq6 and Coq7 hydroxylases, for removal of the nitrogen substituent from pABA-derived Q intermediates, and is an essential component of the CoQ synthome.

N-Acetylation of p-aminobenzoic acid and p-phenylenediamine in primary porcine urinary bladder epithelial cells and in the human urothelial cell line 5637.

N-Acetyltransferases (NAT) are important enzymes in the metabolism of certain carcinogenic arylamines, as N-acetylation decreases or prevents their bioactivation via N-hydroxylation. To study such processes in the bladder, cell culture models may be used, but metabolic competence needs to be characterized. This study focused on the N-acetylation capacity of two urothelial cell systems, using p-aminobenzoic acid (PABA) and the hair dye precursor p-phenylenediamine (PPD), two well-known substrates of the enzyme NAT1. The constitutive NAT1 activity was investigated using primary cultures of porcine urinary bladder epithelial cells (PUBEC) and in the human urothelial cell line 5637 to assess their suitability for further in vitro studies on PABA and PPD-induced toxicity. N-Acetylation of PABA and PPD was determined by high-performance liquid chromatography (HPLC) analysis in cytosols of the two cell systems upon incubation with various substrate levels for up to 60 min. The primary PUBEC revealed higher N-acetylation rates (2.5-fold for PABA, 5-fold for PPD) compared to the 5637 cell line, based on both PABA conversion to its acetylated metabolite and formation of mono- and diacetylated PPD. The urothelial cell systems may thus be useful as a tool for further studies on the N-acetylation of aromatic amines via NAT1.

Cyanamide-mediated Inhibition of N-acetyltransferase 1.

Cyanamide has been used for decades for medical intentions in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. Its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolized in vivo mainly via coenzyme A dependent N-acetylation by N-acetyltransferases. Although described to be a substrate for N-acetyltransferases (NATs), cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for N-acetyltransferases. Therefore, a more detailed investigation of its interrelations with N-acetyltransferases was performed. We analyzed the impact of cyanamide on NAT1 activities of human monocytes (monocytic THP-1 cells) using the classical substrate p-aminobenzoic acid. We found that a 24h treatment with physiologically relevant concentrations of cyanamide decreased the NAT1 activity significantly. Based on this observation we performed additional experiments using recombinant human NAT1 and NAT2 to achieve further insights. In detail a significant dose- and time-dependent inhibition of NAT1 activity was observed for 100 and 1000µM cyanamide using recombinant human NAT1*4. However, cyanamide did not inhibit recombinant NAT2*4. Experiments testing cyanamide as substrate did not provide evidence that cyanamide is metabolized via coenzyme A dependent N-acetylation in vitro by human NAT1 or NAT2, THP-1 or human liver cytosol. Therefore we can conclude that the observed enzyme inhibition (around 50% and 25% after treatment with 0.5 and 0.25mM CA, respectively) is not based on substrate-dependent down-regulation of NAT1. Further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of NAT1, leading to its rapid inhibition (significant inhibition after 30min and 2h for 1000 and 100µM CA, respectively). Addition of the reduction agent dithiothreitol (DTT) did not modify the effect, indicating that oxidative processes that can be reversed by 5mM DTT are not likely involved in the inhibition. Taken together our results show that cyanamide is able to inhibit NAT1 most likely via interaction with the active site cysteine residue. Thereby cyanamide might modulate NAT1 dependent detoxification and activation of arylamines.

Development of a high-affinity inhibitor of the prostaglandin transporter.

Prostaglandin E(2) (PGE(2)) triggers a vast array of biological signals and physiological events. The prostaglandin transporter (PGT) controls PGE(2) influx and is rate-limiting for PGE(2) metabolism and signaling termination. PGT global knockout mice die on postnatal day 1 from patent ductus arteriosus. A high-affinity PGT inhibitor would thus be a powerful tool for studying PGT function in adult animals. Moreover, such an inhibitor could be potentially developed into a therapeutic drug targeting PGT. Based on structure-activity relationship studies that built on recently identified inhibitors of PGT, we obtained N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-4-((4-((2-(2-(2-benzamidoethoxy)ethoxy)ethyl)amino)-6-((4-hydroxyphenyl)amino)-1,3,5-triazin-2-yl)amino)benzamide (T26A), a competitive inhibitor of PGT, with a K(i) of 378 nM. T26A seems to be highly selective for PGT, because it neither interacts with a PGT homolog in the organic anion transporter family nor affects PGE(2) synthesis. In Madin-Darby canine kidney cells stably transfected with PGT, T26A blocked PGE(2) metabolism, resulting in retention of PGE(2) in the extracellular compartment and the negligible appearance of PGE(2) metabolites in the intracellular compartment. Compared with vehicle, T26A injected intravenously into rats effectively doubled the amount of endogenous PGE(2) in the circulation and reduced the level of circulating endogenous PGE(2) metabolites to 50%. Intravenous T26A was also able to slow the metabolism of exogenously injected PGE(2). These results confirm that PGT directly regulates PGE(2) metabolism and demonstrate that a high-affinity inhibitor of PGT can effectively prevent PGE(2) metabolism and prolong the half-life of circulating PGE(2).

Folate production by probiotic bacteria.

Probiotic bacteria, mostly belonging to the genera Lactobacillus and Bifidobacterium, confer a number of health benefits to the host, including vitamin production. With the aim to produce folate-enriched fermented products and/or develop probiotic supplements that accomplish folate biosynthesis in vivo within the colon, bifidobacteria and lactobacilli have been extensively studied for their capability to produce this vitamin. On the basis of physiological studies and genome analysis, wild-type lactobacilli cannot synthesize folate, generally require it for growth, and provide a negative contribution to folate levels in fermented dairy products. Lactobacillus plantarum constitutes an exception among lactobacilli, since it is capable of folate production in presence of para-aminobenzoic acid (pABA) and deserves to be used in animal trials to validate its ability to produce the vitamin in vivo. On the other hand, several folate-producing strains have been selected within the genus Bifidobacterium, with a great variability in the extent of vitamin released in the medium. Most of them belong to the species B. adolescentis and B. pseudocatenulatum, but few folate producing strains are found in the other species as well. Rats fed a probiotic formulation of folate-producing bifidobacteria exhibited increased plasma folate level, confirming that the vitamin is produced in vivo and absorbed. In a human trial, the same supplement raised folate concentration in feces. The use of folate-producing probiotic strains can be regarded as a new perspective in the specific use of probiotics. They could more efficiently confer protection against inflammation and cancer, both exerting the beneficial effects of probiotics and preventing the folate deficiency that is associated with premalignant changes in the colonic epithelia.

N-acetyltransferase 2 (Nat2) polymorphism in the sand rat Psammomys obesus.

Human arylamine N-acetyltransferase 1 (NAT1) and its homologue in rodents (Nat2) are polymorphic xenobiotic metabolizing enzymes and also seem to play a role in endogenous metabolism. NAT1 and Nat2 polymorphism was associated to cancers under xenobiotic procarcinogens metabolism as well as under endogenous substrate metabolism. This study investigated the p-aminobenzoic acid (PABA) -Nat2 catalytic activity and its polymorphism in liver homogenates of adult sand rats Psammomys obesus Cretzschmar, 1828. These Saharian sand rats develop high incidence of spontaneous cancers under standard laboratory diet. The average value of PABA-Nat2 specific activity tested in nine sand rats was significant (2.96 ± 2.16 nmoles/min/mg). The N-acetylation exhibited a bimodal distribution. There was a significant difference (p<0.01) between PABA-Nat2 activity in the fast acetylators group (4.10 ± 1.67 nmol/min/mg) and slow acetylators group (0.7 ± 0.27 nmol/min/mg). The percentage of the fast acetylator group was 66.66%. These results support the presence of Nat2 polymorphism in the liver of the strain sand rats Psammomys obesus. This strain is useful for investigating the role of Nat2 polymorphisms in susceptibility to cancers related to arylamine carcinogen exposures as well as to endogenous substrate metabolism.

Codominant expression of N-acetylation and O-acetylation activities catalyzed by N-acetyltransferase 2 in human hepatocytes.

Human populations exhibit genetic polymorphism in N-acetylation capacity, catalyzed by N-acetyltransferase 2 (NAT2). We investigated the relationship between NAT2 acetylator genotype and phenotype in cryopreserved human hepatocytes. NAT2 genotypes determined in 256 human samples were assigned as rapid (two rapid alleles), intermediate (one rapid and one slow allele), or slow (two slow alleles) acetylator phenotypes based on functional characterization of the NAT2 alleles reported previously in recombinant expression systems. A robust and significant relationship was observed between deduced NAT2 phenotype (rapid, intermediate, or slow) and N-acetyltransferase activity toward sulfamethazine (p < 0.0001) and 4-aminobiphenyl (p < 0.0001) and for O-acetyltransferase-catalyzed metabolic activation of N-hydroxy-4-aminobiphenyl (p < 0.0001), N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (p < 0.01), and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (p < 0.0001). NAT2-specific protein levels also significantly associated with the rapid, intermediate, and slow NAT2 acetylator phenotypes (p < 0.0001). As a negative control, p-aminobenzoic acid (an N-acetyltransferase 1-selective substrate) N-acetyltransferase activities from the same samples did not correlate with the three NAT2 acetylator phenotypes (p > 0.05). These results clearly document codominant expression of human NAT2 alleles resulting in rapid, intermediate, and slow acetylator phenotypes. The three phenotypes reflect levels of NAT2 protein catalyzing both N- and O-acetylation. Our results suggest a significant role of NAT2 acetylation polymorphism in arylamine-induced cancers and are consistent with differential cancer risk and/or drug efficacy/toxicity in intermediate compared with rapid or slow NAT2 acetylator phenotypes.

Skin metabolism of aminophenols: human keratinocytes as a suitable in vitro model to qualitatively predict the dermal transformation of 4-amino-2-hydroxytoluene in vivo.

4-Amino-2-hydroxytolune (AHT) is an aromatic amine ingredient in oxidative hair colouring products. As skin contact occurs during hair dyeing, characterisation of dermal metabolism is important for the safety assessment of this chemical class. We have compared the metabolism of AHT in the human keratinocyte cell line HaCaT with that observed ex-vivo in human skin and in vivo (topical application versus oral (p.o.) and intravenous (i.v.) route). Three major metabolites of AHT were excreted, i.e. N-acetyl-AHT, AHT-sulfate and AHT-glucuronide. When 12.5 mg/kg AHT was applied topically, the relative amounts of each metabolite were altered such that N-acetyl-AHT product was the major metabolite (66% of the dose in comparison with 37% and 32% of the same applied dose after i.v. and p.o. administration, respectively). N-acetylated products were the only metabolites detected in HaCaT cells and ex-vivo whole human skin discs for AHT and p-aminophenol (PAP), an aromatic amine known to undergo N-acetylation in vivo. Since N-acetyltransferase 1 (NAT1) is the responsible enzyme, kinetics of AHT was further compared to the standard NAT1 substrate p-aminobenzoic acid (PABA) in the HaCaT model revealing similar values for K(m) and V(max). In conclusion NAT1 dependent dermal N-acetylation of AHT represents a 'first-pass' metabolism effect in the skin prior to entering the systemic circulation. Since the HaCaT cell model represents a suitable in vitro assay for addressing the qualitative contribution of the skin to the metabolism of topically-applied aromatic amines it may contribute to a reduction in animal testing.

4-Aminobiphenyl downregulation of NAT2 acetylator genotype-dependent N- and O-acetylation of aromatic and heterocyclic amine carcinogens in primary mammary epithelial cell cultures from rapid and slow acetylator rats.

Aromatic and heterocyclic amine carcinogens present in the diet and in cigarette smoke induce breast tumors in rats. N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) enzymes have important roles in their metabolic activation and deactivation. Human epidemiological studies suggest that genetic polymorphisms in NAT1 and/or NAT2 modify breast cancer risk in women exposed to these carcinogens. p-Aminobenzoic acid (selective for rat NAT2) and sulfamethazine (SMZ; selective for rat NAT1) N-acetyltransferase catalytic activities were both expressed in primary cultures of rat mammary epithelial cells. PABA, 2-aminofluorene, and 4-aminobiphenyl N-acetyltransferase and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline O-acetyltransferase activities were two- to threefold higher in mammary epithelial cell cultures from rapid than slow acetylator rats. In contrast, SMZ (a rat NAT1-selective substrate) N-acetyltransferase activity did not differ between rapid and slow acetylators. Rat mammary cells cultured in the medium supplemented 24 h with 10muM ABP showed downregulation in the N-and O-acetylation of all substrates tested except for the NAT1-selective substrate SMZ. This downregulation was comparable in rapid and slow NAT2 acetylators. These studies clearly show NAT2 acetylator genotype-dependent N- and O-acetylation of aromatic and heterocyclic amine carcinogens in rat mammary epithelial cell cultures to be subject to downregulation by the arylamine carcinogen ABP.

Systemic functional expression of N-acetyltransferase polymorphism in the F344 Nat2 congenic rat.

Rat lines congenic for the rat N-acetyltransferase 2 [(RAT)Nat2] gene were constructed and characterized. F344 (homozygous Nat2 rapid) males were mated to Wistar Kyoto (homozygous Nat2 slow) females to produce heterozygous F1. F1 females were then backcrossed to F344 males. Heterozygous acetylator female progeny from this and each successive backcross were identified by rat Nat2 genotyping and mated with F344 rapid acetylator males. After 10 generations of backcross mating, heterozygous acetylator brother/sister progeny were mated to produce the homozygous rapid and slow acetylator Nat2 congenic rat lines. p-Aminobenzoic acid (selective for rat NAT2) and 4-aminobiphenyl N-acetyltransferase activities were expressed in all tissues examined (liver, lung, esophagus, stomach, small intestine, colon, pancreas, kidney, skin, leukocytes, and urinary bladder in male and female rats and in breast of female and prostate of male rats). NAT2 expression in rat extrahepatic tissues was much higher than that in liver. In each tissue, activities were Nat2-genotype-dependent, with the highest levels in homozygous rapid acetylators, intermediate levels in heterozygous acetylators, and lowest in homozygous slow acetylators. Sulfamethazine (selective for rat NAT1) N-acetyltransferase activities were observed in all tissues examined in both male and female rats except for breast (females), bladder, and leukocytes. In each tissue, the activity was Nat2 genotype-independent, with similar levels in homozygous rapid, heterozygous, and homozygous slow acetylators. These congenic rat lines are useful for investigating the role of NAT2 genetic polymorphisms in susceptibility to cancers related to arylamine carcinogen exposures.

Laccase-induced C-N coupling of substituted p-hydroquinones with p-aminobenzoic acid in comparison with known chemical routes.

Fungal laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) from Pycnoporus cinnabarinus and Myceliophthora thermophila were used as biocatalysts for enzymatic reaction of halogen-, alkyl-, alkoxy-, and carbonyl-substituted p-hydroquinones (laccase substrates) with p-aminobenzoic acid (no laccase substrate). During this reaction, the laccase substrate was oxidized to the corresponding quinones, which react with p-aminobenzoic acid by amination of the laccase substrate. The different substitutions at the hydroquinone substrates were used to prove whether the substituents influence the position of amination and product yields. The cross-coupling of methoxy-p-hydroquinone (alkoxylated) and 2,5-dihydroxybenzaldehyd (carbonyl-substituted) with p-aminobenzoic acid resulted in the formation of one monoaminated product (yield alkoxylated 52%). If monohalogen- or monoalkyl-substituted p-hydroquinones were used as laccase substrates, two monoaminated products (constitution isomers) were formed. The simultaneous formation of two different monoaminated products from the same hydroquinone substrate is the first report for laccase-mediated synthesis of aminated constitution isomers. Depending from the type of substituent of the hydroquinone, the positions of the two monoaminations are different. While the amination at the monoalkylated hydroquinone occurs at the 5- and 6-positions (yield 38%), the amination at monohalogenated hydroquinones was detectable at the 3- and 5-positions (yield 53%). The same product pattern could be achieved if instead of the biocatalyst laccase the chemical catalyst sodium iodate was used as the oxidant. However, the yields were partially much lower (0-45% of the yields with laccase).

[Metalloproteinases. Structure and function]. / Metaloproteinazy MMP. Struktura i funkcja.

Matrix metalloproteinases (MMPs) belong to a large family of multidomain zinc endopeptidases. They are one of the most important proteolitic enzymes which digest components of the extracellular matrix and abundant macromolecules on cell surface and take part in many physiological processes, such as apoptosis or angiogenesis. MMPs are also engaged in the pathogenesis of many diseases such as arthritis and cancer. The development of effective inhibitors and discovery of their mechanisms of action can have significant influence on therapeutic strategy.

Anticancer drug discovery targeting DNA hypermethylation.

DNA methyltransferases (DNMTs) are important regulators of gene transcription and their roles in carcinogenesis have been a topic of considerable interest in the last few years. Diverse classes of chemical compounds including nucleotide analogues, adenosine analogues, aminobenzoic derivatives, polyphenols, hydrazines, phthalides, disulfides and antisenses are being discovered and evaluated as DNMT inhibitors targeting DNA hypermethylation. Among them, 5-Azacytidine 5 and Decitabine 6 were launched recently. Several other compounds are under clinical trials. Some of these compounds were discovered from structure-based drug design. These compounds exert their DNA methylation inhibitory by different mechanisms. This review will present a brief account of various DNA methyltransferases and their biological functions, with focus on actuality of design and synthesis of various inhibitors of DNA hypermethylation as anticancer drugs.