Propolis alleviates 4-aminobiphenyl-induced oxidative DNA damage by inhibition of CYP2E1 expression in human liver cells.

4-Aminobiphenyl (4-ABP) may cause DNA damage in human liver cells (HepG2 and L-02). Propolis exhibits antioxidant properties through reactive oxygen species (ROS) scavenging. We determined the effects of propolis in alleviating 4-ABP -induced DNA damage using the comet assay. Results revealed that propolis could significantly alleviated oxidative damaged DNA by 4-ABP. Furthermore, we proved that inhibition of cytochrome P450 2E1 (CYP2E1) expression by propolis could contribute to the decreased oxidative DNA damage in the treated cells, as the conversion of 4-ABP into its metabolite, N-hydroxy-ABP (HOABP), was blocked; after all, HOABP showed more genotoxic than its parent chemical, 4-ABP. With the homologous recombination assay, propolis failed to induce DNA repair enzymes. Furthermore, the expression of RAD51, Ku70/Ku80, and OGG1 in treated cells were determined with the western blot, revealing that the expression of these protein were unchanged in comparison with those in nontreated cells. However, propolis could protect the treated cells from DNA damage. In conclusion, propolis could antagonize 4-ABP-induced oxidative DNA damage though the removal of ROS and inhibition of CYP2E1 expression in the treated cells.

RSK-3 promotes cartilage regeneration via interacting with rpS6 in cartilage stem/progenitor cells.

Rationale: Cartilage stem/progenitor cells (CSPC) are a promising cellular source to promote endogenous cartilage regeneration in osteoarthritis (OA). Our previous work indicates that ribosomal s6 kinase 3 (RSK-3) is a target of 4-aminobiphenyl, a chemical enhancing CSPC-mediated cartilage repair in OA. However, the primary function and mechanism of RSK-3 in CSPC-mediated cartilage pathobiology remain undefined. Methods: We systematically assessed the association of RSK-3 with OA in three mouse strains with varying susceptibility to OA (MRL/MpJ>CBA>STR/Ort), and also RSK-3-/- mice. Bioinformatic analysis was used to identify the possible mechanism of RSK-3 affecting CSPC, which was further verified in OA mice and CSPC with varying RSK-3 expression induced by chemicals or gene modification. Results: We demonstrated that the level of RSK-3 in cartilage was positively correlated with cartilage repair capacities in three mouse strains (MRL/MpJ>CBA>STR/Ort). Enhanced RSK-3 expression by 4-aminobiphenyl markedly attenuated cartilage injury in OA mice and inhibition or deficiency of RSK-3 expression, on the other hand, significantly aggravated cartilage damage. Transcriptional profiling of CSPC from mice suggested the potential role of RSK-3 in modulating cell proliferation. It was further shown that the in vivo and in vitro manipulation of the RSK-3 expression indeed affected the CSPC proliferation. Mechanistically, ribosomal protein S6 (rpS6) was activated by RSK-3 to accelerate CSPC growth. Conclusion: RSK-3 is identified as a key regulator to enhance cartilage repair, at least partly by regulating the functionality of the cartilage-resident stem/progenitor cells.

4-Aminobiphenyl inhibits the DNA homologous recombination repair in human liver cells: The role of miR-630 in downregulating RAD18 and MCM8.

4-Aminobiphenyl (4-ABP), a well-known human carcinogen, has been shown to cause oxidative DNA damage and induce miR-630 expression in HepG2 cells treated with 18.75 µM-300 µM for 24 h. However, the underlying mechanism regarding the epigenetic regulation of miR-630 on DNA damage repair in liver cells is still not understood and needs to be investigated. In present study, our results showed that miR-630 was upregulated, resulting in mediating a decrease of DNA homologous recombination (HR) repair in L-02, HepG2 or Hep3B cells. Results from a luciferase reporting experiment showed that RAD18 and MCM8 were the potential targets of miR-630 during DNA damage induction. The downregulation of RAD18 or MCM8 by miR-630 was accompanied by inhibition of HR repair. Conversely, inhibiting miR-630 enhanced the expression of RAD18 and MCM8, and rescued HR repair. Additionally, we proved that the transcription factor CREB was related to miR-630 biogenesis in liver cells. Moreover, the levels of CREB, miR-630 expression, and double-strand breaks (DSBs) were attenuated by 5 mM N-acetyl-L-cysteine (NAC) pretreatment, indicating that reactive oxygen species (ROS)-dependent CREB-miR-630 was involved in DSB repair. These findings indicated that the ROS/CREB/-miR-630 axis plays a relevant role in the regulation of RAD18 and MCM8 in HR repair, which may facilitate our understanding of molecular mechanisms regarding the role of miR-630 downregulating DNA damage repair in liver cells.

Regiospecific Introduction of Halogens on the 2-Aminobiphenyl Subunit Leading to Highly Potent and Selective M3 Muscarinic Acetylcholine Receptor Antagonists and Weak Inverse Agonists.

Muscarinic M3 receptor antagonists and inverse agonists displaying high affinity and subtype selectivity over the antitarget M2 are valuable pharmacological tools and may enable improved treatment of chronic obstructive pulmonary disease (COPD), asthma, or urinary incontinence. On the basis of known M3 antagonists comprising a piperidine or quinuclidine unit attached to a biphenyl carbamate, 5-fluoro substitution was responsible for M3 subtype selectivity over M2, while 3'-chloro substitution substantially increased affinity through a σ-hole interaction. Resultantly, two piperidinyl- and two quinuclidinium-substituted biphenyl carbamates OFH243 (13n), OFH244 (13m), OFH3911 (14n), and OFH3912 (14m) were discovered, which display two-digit picomolar affinities with Ki values from 0.069 to 0.084 nM, as well as high selectivity over the M2 subtype (46- to 68-fold). While weak inverse agonistic properties were determined for the biphenyl carbamates 13m and 13n, neutral antagonism was observed for 14m and 14n and tiotropium under identical assay conditions.

Evaluation of biophysical as well as biochemical potential of curcumin and resveratrol during prostate cancer.

Purpose: The present study evaluated biochemical as well as biophysical mechanisms behind the synergistic effects of curcumin and resveratrol during prostate carcinogenesis.Methods: The rats were segregated into five groups that included normal control, 3,2'-dimethyl-4-aminobiphenyl (DMAB)treated, DMAB + curcumin treated, DMAB + resveratrol-treated and DMAB + curcumin + resveratrol-treated.Results: The DMAB treatment resulted in a significant increase in the levels of lipid peroxidation (LPO) in DMAB treated rats. Also, significant changes were recorded in the enzyme activities of both drug metabolising enzyme and antioxidant enzymes after DMAB treatment. Further, radiorespirometric studies showed a significant increase in the 14C-glucose turnover as well as 14C-glucose uptake in the prostate slices of DMAB treated rats. Moreover, a significant rise in cell proliferation was confirmed indirectly by enhanced uptake of 3H-thymidine in the prostate slices of DMAB treated rats. Interestingly, combined treatment of curcumin and resveratrol to DMAB treated animals resulted in a significant decrease in lipid peroxidation, 14C glucose uptakes/turnover and 3H-thymidine uptake in the DMAB treated rats. Besides this, curcumin and resveratrol in combination significantly modulated biochemical indices including drug-metabolising enzymes; antioxidant enzymes in DMBA treated rats.Conclusion: The study, therefore, concludes that the combination of curcumin and resveratrol holds strong modulatory potential against prostate carcinogenesis.

N-hydroxylation of 4-aminobiphenyl by CYP2E1 produces oxidative stress in a mouse model of chemically induced liver cancer.

4-Aminobiphenyl (ABP) is a trace component of cigarette smoke and hair dyes, a suspected human carcinogen and a potent rodent liver carcinogen. Postnatal exposure of mice to ABP results in a higher incidence of liver tumors in males than in females, paralleling the sex difference in human liver cancer incidence. A traditional model of ABP tumorigenesis involves initial CYP1A2-mediated N-hydroxylation, which eventually leads to production of mutagenic ABP-DNA adducts that initiate tumor growth. However, several studies have found no correlation between sex or CYP1A2 function and the DNA-damaging, mutagenic, or tumorigenic effects of ABP. Oxidative stress may be an important etiological factor for liver cancer, and it has also been linked to ABP exposure. The goals of this study were to identify novel enzyme(s) that contribute to ABP N-oxidation, and to investigate a potential role for oxidative stress in ABP liver tumorigenicity. Isozyme-selective inhibition experiments using liver microsomes from wild-type and genetically modified mice identified CYP2E1 as a major ABP N-hydroxylating enzyme. The N-hydroxylation of ABP by transiently expressed CYP2E1 produced oxidative stress in cultured mouse hepatoma cells. In vivo postnatal exposure of mice to a tumorigenic dose of ABP also produced oxidative stress in male wild-type mice, but not in male Cyp2e1(-/-) mice or in female mice. However, a stronger NRF2-associated antioxidant response was observed in females. Our results identify CYP2E1 as a novel ABP-N-oxidizing enzyme, and suggest that sex differences in CYP2E1-dependent oxidative stress and antioxidant responses to ABP may contribute to the observed sex difference in tumor incidence.

DNA adducts of the tobacco carcinogens 2-amino-9H-pyrido[2,3-b]indole and 4-aminobiphenyl are formed at environmental exposure levels and persist in human hepatocytes.

Aromatic amines and structurally related heterocyclic aromatic amines (HAAs) are produced during the combustion of tobacco or during the high-temperature cooking of meat. Exposure to some of these chemicals may contribute to the etiology of several common types of human cancers. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant HAA formed in mainstream tobacco smoke: it arises in amounts that are 25-100 times greater than the levels of the arylamine, 4-aminobiphenyl (4-ABP), a human carcinogen. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a prevalent HAA formed in cooked meats. AαC and MeIQx are rodent carcinogens; however, their carcinogenic potency in humans is unknown. A preliminary assessment of the carcinogenic potential of these HAAs in humans was conducted by examining the capacity of primary human hepatocytes to form DNA adducts of AαC and MeIQx, in comparison to 4-ABP, followed by the kinetics of DNA adduct removal by cellular enzyme repair systems. The principal DNA adducts formed were N-(deoxyguanosin-8-yl) (dG-C8) adducts. Comparable levels of DNA adducts were formed with AαC and 4-ABP, whereas adduct formation was ∼5-fold lower for MeIQx. dG-C8-AαC and dG-C8-4-ABP were formed at comparable levels in a concentration-dependent manner in human hepatocytes treated with procarcinogens over a 10,000-fold concentration range (1 nM-10 µM). Pretreatment of hepatocytes with furafylline, a selective inhibitor of cytochrome P450 1A2, resulted in a strong diminution of DNA adducts signifying that P450 1A2 is a major P450 isoform involved in bioactivation of these procarcinogens. The kinetics of adduct removal varied for each hepatocyte donor. Approximately half of the DNA adducts were removed within 24 h of treatment; however, the remaining lesions persisted over 5 days. The high levels of AαC present in tobacco smoke and its propensity to form persistent DNA adducts in human hepatocytes suggest that AαC can contribute to DNA damage and the risk of hepatocellular cancer in smokers.

Functional analysis of arylamine N-acetyltransferase 1 (NAT1) NAT1*10 haplotypes in a complete NATb mRNA construct.

N-acetyltransferase 1 (NAT1) catalyzes N-acetylation of arylamines as well as the O-acetylation of N-hydroxylated arylamines. O-acetylation leads to the formation of electrophilic intermediates that result in DNA adducts and mutations. NAT1*10 is the most common variant haplotype and is associated with increased risk for numerous cancers. NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in messenger RNAs (mRNAs) with distinct 5'-untranslated regions (UTRs). To best mimic in vivo metabolism and the effect of NAT1*10 polymorphisms on polyadenylation usage, pcDNA5/Flp recombination target plasmid constructs were prepared for transfection of full-length human mRNAs including the 5'-UTR derived from NATb, the open reading frame and 888 nucleotides of the 3'-UTR. Following stable transfection of NAT1*4, NAT1*10 and an additional NAT1*10 variant (termed NAT1*10B) into nucleotide excision repair-deficient Chinese hamster ovary cells, N- and O-acetyltransferase activity (in vitro and in situ), mRNA and protein expression were higher in cells transfected with NAT1*10 and NAT1*10B than in cells transfected with NAT1*4 (P < 0.05). Consistent with NAT1 expression and activity, cytotoxicity and hypoxanthine phosphoribosyl transferase mutants following 4-aminobiphenyl exposures were higher in NAT1*10 than in NAT1*4 transfected cells. Ribonuclease protection assays showed no difference between NAT1*4 and NAT1*10. However, protection of one probe by NAT1*10B was not observed with NAT1*4 or NAT1*10, suggesting additional mechanisms that regulate NAT1*10B. The higher mutants in cells transfected with NAT1*10 and NAT1*10B are consistent with an increased cancer risk for individuals possessing NAT1*10 haplotypes.

Sulforaphane inhibits 4-aminobiphenyl-induced DNA damage in bladder cells and tissues.

Sulforaphane (SF) is a well-known chemopreventive phytochemical and occurs in broccoli and to a lesser extent in other cruciferous vegetables, whereas 4-aminobiphenyl (ABP) is a major human bladder carcinogen and is present at significant levels in tobacco smoke. Here, we show that SF inhibits ABP-induced DNA damage in both human bladder cells in vitro and mouse bladder tissue in vivo, using dG-C8-ABP as a biomarker, which is the predominant ABP-DNA adduct formed in human bladder cells and tissues. SF activates NF-E2 related factor-2 (Nrf2), which is a well-recognized chemopreventive target and activates the Nrf2-regulated cytoprotective signaling pathway. Comparison between wild-type mice and mice without Nrf2 shows that Nrf2 activation is required by SF for inhibition of ABP-induced DNA damage. Moreover, Nrf2 activation by SF in the bladder occurs primarily in the epithelium, which is the principal site of bladder cancer development. These data, together with our recent observation that SF-enriched broccoli sprout extracts strongly inhibits N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer development, suggest that SF is a highly promising agent for bladder cancer prevention and provides a mechanistic insight into the repeated epidemiological observation that consumption of broccoli is inversely associated with bladder cancer risk and mortality.

Differences between human slow N-acetyltransferase 2 alleles in levels of 4-aminobiphenyl-induced DNA adducts and mutations.

Aromatic amines such as 4-aminobiphenyl (ABP) require biotransformation to exert their carcinogenic effects. Genetic polymorphisms in biotransformation enzymes such as N-acetyltransferase 2 (NAT2) may modify cancer risk following exposure. Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator), NAT2*5B (common Caucasian slow acetylator), or NAT2*7B (common Asian slow acetylator) alleles (haplotypes) were treated with ABP to test the effect of NAT2 polymorphisms on DNA adduct formation and mutagenesis. ABP N-acetyltransferase catalytic activities were detectable only in cell lines transfected with NAT2 and were highest in cells transfected with NAT2*4, lower in cells transfected with NAT2*7B, and lowest in cells transfected with NAT2*5B. Following ABP treatment, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) was the primary adduct formed. Cells transfected with both CYP1A1 and NAT2*4 showed the highest concentration-dependent cytotoxicity, hypoxanthine phosphoribosyl transferase (hprt) mutants, and dG-C8-ABP adducts. Cells transfected with CYP1A1 and NAT2*7B showed lower levels of cytotoxicity, hprt mutagenesis, and dG-C8-ABP adducts. Cells transfected with CYP1A1 only or cells transfected with both CYP1A1 and NAT2*5B did not induce cytotoxicity, hprt mutagenesis or dG-C8-ABP adducts. ABP-DNA adduct levels correlated very highly (r>0.96) with ABP-induced hprt mutant levels following each treatment. The results of the present study suggest that investigations of NAT2 genotype or phenotype associations with disease or toxicity could be more precise and reproducible if heterogeneity within the "slow" NAT2 acetylator phenotype is considered and incorporated into the study design.

Effects of endocrine disrupting chemicals from leather industry effluents on male reproductive system.

The leather tanning industry is characterized by the production of different kinds of effluents, generated in each step of leather processing. These effluents have various chemical compounds which may cause toxicity and endocrine disruption and are thus known as endocrine disrupting chemicals (EDC). This study was aimed to examine the androgenic potential of leather industry effluents collected from northern region of India. Hershberger assay data showed a significant increase (p<0.05) in the weight and structure of sex accessory tissues of castrated rats. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis demonstrated a significant change (p<0.05) in the expression patterns of the major steroidogenic enzymes in adrenal and testes namely, cytochrome P450scc, 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydorgenase in castrated and intact rats. This was further supported by increased enzymatic activities measured in vitro spectrophotometrically. Serum hormone profile demonstrated a dose dependent increase in testicular and adrenal testosterone productions in intact and castrated rats, respectively. This was further supported by decreased level of gonadotrophic hormones (LH and FSH) in treated groups of animals. Further, the effluent treatment resulted in the development of hyperplasia in seminiferous tubules of testes in treated rats as evident from histopathological studies and about two-fold increases in daily sperm production. On analysis of water samples using GC-MS, it was found to contain various aromatic compounds (nonylphenol, hexaclrobenzene and several azo dyes) some of which independently demonstrated similar effects as shown by water samples. Our data suggests that the effluents from leather industry have potential EDC demonstrating androgenic activities.

Pyrimidine nucleoside depletion sensitizes to the mitochondrial hepatotoxicity of the reverse transcriptase inhibitor stavudine.

Stavudine is a hepatotoxic antiretroviral nucleoside analogue that also inhibits the replication of mitochondrial DNA (mtDNA). To elucidate the mechanism and consequences of mtDNA depletion, we treated HepG2 cells with stavudine and either redoxal, an inhibitor of de novo pyrimidine synthesis, or uridine, from which pyrimidine pools are salvaged. Compared with treatment with stavudine alone, co-treatment with redoxal accelerated mtDNA depletion, impaired cell division, and activated caspase 3. These adverse effects were completely abrogated by uridine. Intracellular ATP levels were unaffected. Transcriptosome profiling demonstrated that redoxal and stavudine acted synergistically to induce CDKN2A and p21, indicating cell cycle arrest in G1, as well as genes involved in intrinsic and extrinsic apoptosis. Moreover, redoxal and stavudine showed synergistic interaction in the up-regulation of transcripts encoded by mtDNA and the induction of nuclear transcripts participating in energy metabolism, mitochondrial biogenesis, oxidative stress, and DNA repair. Genes involved in nucleotide metabolism were also synergistically up-regulated by both agents; this effect was completely antagonized by uridine. Thus, pyrimidine depletion sensitizes cells to stavudine-mediated mtDNA depletion and enhances secondary cell toxicity. Our results indicate that drugs that diminish pyrimidine pools should be avoided in stavudine-treated human immunodeficiency virus patients. Uridine supplementation reverses this toxicity and, because of its good tolerability, has potential clinical value for the treatment of side effects associated with pyrimidine depletion.

Novel aminoethylbiphenyls as 5-HT7 receptor ligands.

The synthesis of a series of aminoethylbiphenyls as novel 5-HT(7) receptor ligands is described. The novel derivatives exhibit high affinity for the 5-HT(7) receptor with selectivity toward 5-HT(1A) receptor.

Cigarette smoke toxicants as substrates and inhibitors for human cytosolic SULTs.

The current study was designed to examine the role of sulfation in the metabolism of cigarette smoke toxicants and clarify whether these toxicants, by serving as substrates for the cytosolic sulfotransferases (SULTs), may interfere with the sulfation of key endogenous compounds. By metabolic labeling, [(35)S]sulfated species were found to be generated and released into the media of HepG2 human hepatoma cells and primary human lung endothelial cells labeled with [(35)S]sulfate in the presence of cigarette smoke extract (CSE). Concomitantly, several [(35)S]sulfated metabolites observed in the medium in the absence of CSE either decreased or disappeared. Eleven previously prepared human cytosolic SULTs were tested for sulfating activity with CSE and known cigarette smoke toxicants as substrates. Activity data revealed SULT1A1, SULT1A2, SULT1A3, and SULT1C#2 as major enzymes responsible for their sulfation. To examine their inhibitory effects on the sulfation of 17beta-estradiol by SULT1A1, enzymatic assays were performed in the presence of three representative toxicant compounds, namely N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), 4-aminobiphenyl (4-ABP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). IC(50) values determined for the sulfation of 17beta-estradiol by SULT1A1 were 11.8 microM, 28.2 microM, and 500 microM, respectively, for N-OH-4-ABP, 4-ABP and PhIP. Kinetic analyses indicated that the mechanism underlying the inhibition of 17beta-estradiol sulfation by these cigarette smoke toxicants is of a mixed competitive-noncompetitive type. Metabolic labeling experiments clearly showed inhibition of the production of [(35)S]sulfated 17beta-estradiol by N-OH-4-ABP in a concentration-dependent manner in HepG2 cells. Taken together, these results suggest that sulfation plays a significant role in the metabolism of cigarette smoke compounds. By serving as substrates for SULTs, cigarette smoke toxicants may interfere with the metabolism of 17beta-estradiol and other endogenous compounds.

Design and synthesis of potent inhibitors of the malaria parasite dihydroorotate dehydrogenase.

Pyrimidine biosynthesis presents an attractive drug target in malaria parasites due to the absence of a pyrimidine salvage pathway. A set of compounds designed to inhibit the Plasmodium falciparum pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (PfDHODH) was synthesized. PfDHODH-specific inhibitors with low nanomolar binding affinities were identified that bind in the N-terminal hydrophobic channel of dihydroorotate dehydrogenase, the presumed site of ubiquinone binding during oxidation of dihydroorotate to orotate. These compounds also prevented growth of cultured parasites at low micromolar concentrations. Models that suggest the mode of inhibitor binding is based on shape complementarity, matching hydrophobic regions of inhibitor and enzyme, and interaction of inhibitors with amino acid residues F188, H185, and R265 are supported by mutagenesis data. These results further highlight PfDHODH as a promising new target for chemotherapeutic intervention in prevention of malaria and provide better understanding of the factors that determine specificity over human dihydroorotate dehydrogenase.

Development of orally bioavailable and CNS penetrant biphenylaminocyclopropane carboxamide bradykinin B1 receptor antagonists.

A series of biphenylaminocyclopropane carboxamide based bradykinin B1 receptor antagonists has been developed that possesses good pharmacokinetic properties and is CNS penetrant. Discovery that the replacement of the trifluoropropionamide in the lead structure with polyhaloacetamides, particularly a trifluoroacetamide, significantly reduced P-glycoprotein mediated efflux for the series proved essential. One of these novel bradykinin B1 antagonists (13b) also exhibited suitable pharmacokinetic properties and efficient ex vivo receptor occupancy for further development as a novel approach for the treatment of pain and inflammation.

4-Aminobiphenyl and DNA reactivity: case study within the context of the 2006 IPCS Human Relevance Framework for Analysis of a cancer mode of action for humans.

The IPCS Human Relevance Framework was evaluated for a DNA-reactive (genotoxic) carcinogen, 4-aminobiphenyl, based on a wealth of data in animals and humans. The mode of action involves metabolic activation by N-hydroxylation, followed by N-esterification leading to the formation of a reactive electrophile, which binds covalently to DNA, principally to deoxyguanosine, leading to an increased rate of DNA mutations and ultimately to the development of cancer. In humans and dogs, the urinary bladder urothelium is the target organ, whereas in mice it is the bladder and liver; in other species, other tissues can be involved. Differences in organ specificity are thought to be due to differences in metabolic activation versus inactivation. Based on qualitative and quantitative considerations, the mode of action is possible in humans. Other biological processes, such as toxicity and regenerative proliferation, can significantly influence the dose response of 4-aminobiphenyl-induced tumors. Based on the IPCS Human Relevance Framework, 4-aminobiphenyl would be predicted to be a carcinogen in humans, and this is corroborated by extensive epidemiologic evidence. The IPCA Human Relevance Framework is useful in evaluating DNA-reactive carcinogens.

Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans.

Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full-length DHODH and N-terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.

Levels of H-ras codon 61 CAA to AAA mutation: response to 4-ABP-treatment and Pms2-deficiency.

DNA mismatch repair (MMR) deficiencies result in increased frequencies of spontaneous mutation and tumor formation. In the present study, we tested the hypothesis that a chemically-induced mutational response would be greater in a mouse with an MMR-deficiency than in the MMR-proficient mouse models commonly used to assay for chemical carcinogenicity. To accomplish this, the induction of H-ras codon 61 CAA-->AAA mutation was examined in Pms2 knockout mice (Pms2-/-, C57BL/6 background) and sibling wild-type mice (Pms2+/+). Groups of five or six neonatal male mice were treated with 0.3 micromol 4-aminobiphenyl (4-ABP) or the vehicle control, dimethylsulfoxide. Eight months after treatment, liver DNAs were isolated and analysed for levels of H-ras codon 61 CAA-->AAA mutation using allele-specific competitive blocker-PCR. In Pms2-proficient and Pms2-deficient mice, 4-ABP treatment caused an increase in mutant fraction (MF) from 1.65x10(-5) to 2.91x10(-5) and from 3.40x10(-5) to 4.70x10(-5), respectively. Pooling data from 4-ABP-treated and control mice, the approximately 2-fold increase in MF observed in Pms2-deficient as compared with Pms2-proficient mice was statistically significant (P=0.0207) and consistent with what has been reported previously in terms of induction of G:C-->T:A mutation in a Pms2-deficient background. Pooling data from both genotypes, the increase in H-ras MF in 4-ABP-treated mice, as compared with control mice, did not reach the 95% confidence level of statistical significance (P=0.0606). The 4-ABP treatment caused a 1.76-fold and 1.38-fold increase in average H-ras MF in Pms2-proficient and Pms2-deficient mice, respectively. Furthermore, the levels of induced mutation in Pms2-proficient and Pms2-deficient mice were nearly identical (1.26x10(-5) and 1.30x10(-5), respectively). We conclude that Pms2-deficiency does not result in an amplification of the H-ras codon 61 CAA-->AAA mutational response induced by 4-ABP.

Effect of inhibitors of histone deacetylase on the induction of cell differentiation in murine and human erythroleukemia cell lines.

Histone deacetylase (HDAC) inhibitors are a novel class of promising anti-cancer agents. Little information is available on the capacity of structurally different HDAC inhibitors to induce terminal cell differentiation in different cell types in relation to enzyme inhibition and subtype selectivity. Consequently, the aim of this study was to provide a comprehensive comparison of these effects. New biarylalanine inhibitors of HDAC were synthesized and compared to a series of standard inhibitors from different laboratories. Chromatographically purified rat liver and immunoprecipitated FLAG-tagged recombinant human HDACs were used as sources of HDAC activity. Enzyme inhibition was studied using a fluorescent substrate and its conversion was monitored by high-performance liquid chromatography. The ability to induce cell differentiation was compared in murine (Friend DS-19) and human (K562) erythroleukemic cell lines, and was quantified by benzidine staining. Inhibition of cell proliferation was evaluated by cell counting. All HDAC inhibitors were identified as potent inhibitors of erythroleukemic cell proliferation. However, we observed a complex pattern of differentiation induction: structurally similar inhibitors resulted in disparate activity profiles, whereas similar profiles were detected within distinct structural classes. Among the newly synthesized biarylalanine compounds, a 3'-methoxy derivative was identified as a very effective inducer of terminal cell differentiation. We conclude that investigation of subtype selectivity of selected HDAC inhibitors does not provide a clear link between selectivity and the observed cellular activity profile. The predictive value of in vitro HDAC inhibition assays for identifying anti-proliferative compounds has been emphasized.