The metabolism of formyl-substituted benzanthracenes to hydroxymethyl metabolites in rat liver in vitro and in vivo

Hydroxylation of benzylic methyl carbon atoms on drugs and carcinogenic polycyclic aromatic hydrocarbons (PAHs) forms benzylic alcohols. Many carcinogenic and mutagenic PAHs bear a primary or secondary benzylic hydroxyl group attached to the meso-region of the molecule. According to the unified theory, PAHs bearing a benzylic hydroxyl group are proximate carcinogenic metabolites. This paper demonstrates that carcinogenic benz[a]anthracenes bearing a formyl group at the meso-region undergo enzymatic reductive metabolism to the corresponding carcinogenic benzylic alcohol in vitro and in vivo. The unified theory would then predict sulfuric acid esterification of such benzylic alcohols as the final common step in their metabolic activation to generate ultimate electrophilic benzylic carbocations. Finally, oxidative metabolism of 7-formylbenz[a]anthracenes gives rise to corresponding carboxylic acids and other oxygenated metabolites that are carcinogenically inert. Thus, oxidative metabolism of meso-region formyl compounds represents an avenue for the elimination of the carcinogen in a detoxified form.     

Modeling of the mechanism of nucleophilic aromatic substitution of fungicide chlorothalonil by glutathione

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile, TCIN, CAS 1897-45-6) is a broad range spectrum fungicide whose fungitoxic action has been associated with the rapid formation of conjugated chlorothalonil–cellular thiol derivatives, specifically with thiol-rich enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and with glutathione (GSH). The biotransformation reaction sequence between enzyme-activated glutathione (GSH) and chlorothalonil depletes cellular glutathione reserves. The conjugation of glutathione with chlorothalonil via nucleophilic aromatic substitution was modeled for an isolated reacting species using semiempirical self-consistent field molecular orbital (SCF-MO) theory at the PM3 level. The potential energy hypersurface at each of the three possible chlorinated attack sites on chlorothalonil was elaborated using a thiolate (CH₃S^–) anion as a model for an enzyme-activated glutathione molecule. Calculated free energies of activation for formation of mono-RSH conjugates suggest that the order of nucleophilic attack on chlorine positions in TCIN is 2>4, 6>5 although energy differences are small (on the order of 1–2 kcal mol^–1). Meisenheimer or -complexes have been isolated as true intermediates on the hypersurface for each reaction, suggesting that the mechanism follows a two-step pathway.Electronic Supplementary Material available.     

The role of aromatic residues in the hydrophobic core of the villin headpiece subdomain

Small autonomously folding proteins are of interest as model systems to study protein folding, as the same molecule can be used for both experimental and computational approaches. The question remains as to how well these minimized peptide model systems represent larger native proteins. For example, is the core of a minimized protein tolerant to mutation like larger proteins are? Also, do minimized proteins use special strategies for specifying and stabilizing their folded structure? Here we examine these questions in the 35‐residue autonomously folding villin headpiece subdomain (VHP subdomain). Specifically, we focus on a cluster of three conserved phenylalanine (F) residues F47, F51, and F58, that form most of the hydrophobic core. These three residues are oriented such that they may provide stabilizing aromatic–aromatic interactions that could be critical for specifying the fold. Circular dichroism and 1D‐NMR spectroscopy show that point mutations that individually replace any of these three residues with leucine were destabilized, but retained the native VHP subdomain fold. In pair‐wise replacements, the double mutant that retains F58 can adopt the native fold, while the two double mutants that lack F58 cannot. The folding of the double mutant that retains F58 demonstrates that aromatic–aromatic interactions within the aromatic cluster are not essential for specifying the VHP subdomain fold. The ability of the VHP subdomain to tolerate mutations within its hydrophobic core indicates that the information specifying the three dimensional structure is distributed throughout the sequence, as observed in larger proteins. Thus, the VHP subdomain is a legitimate model for larger, native proteins.     

A site-specific mutation of tyrosine hydroxylase reduces feedback inhibition by dopamine in genetically modified cells grafted in parkinsonian rats

Aromatic L-amino acid decarboxylase (AADC) is necessary for conversion of L-DOPA to dopamine. Therefore, AADC gene therapy has been proposed to enhance pharmacological or gene therapies delivering L-DOPA. However, addition of AADC to the grafts of genetically modified cells expressing tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1), which produce L-DOPA in parkinsonian rats, resulted in decreased production of L-DOPA and dopamine owing to feedback inhibition of TH by dopamine. End-product feedback inhibition has been shown to be mediated by the regulatory domain of TH, and site-specific mutation of serine 40 makes TH less susceptible to dopamine inhibition. Therefore, we investigated the efficacy of using TH with serine 40 mutated to leucine (mTH) in an ex vivo gene-therapy paradigm. Primary fibroblasts (PF) from Fischer 344 rats were transduced with retrovirus to express mTH or wild-type rat TH cDNA (wtTH). Both cell types were also transduced with GCH1 to provide the obligate TH cofactor, tetrahydrobiopterin. PF transfected with AADC were used as coculture and cografting partners. TH activities and L-DOPA production in culture were comparable between PFwtTHGC and PFmTHGC cells. In cocultures with PFAADC cells, PFmTHGC cells showed significant reduction in the inhibitory effect of dopamine compared with PFwtTHGC cells. In vivo microdialysis measurement showed that cografting PFAADC cells with PFmTHGC cells resulted in smaller decreases in L-DOPA and no reduction in dopamine levels compared with cografts of PFAADC cells with PFwtTHGC cells, which decreased both L-DOPA and dopamine levels. Maintenance of dopamine levels with lower levels of L-DOPA would result in more focused local delivery of dopamine and less potential side-effects arising from L-DOPA diffusion into other structures. These data support the hypothesis that mutation of serine 40 attenuates TH end-product inhibition in vivo and illustrates the importance of careful consideration of biochemical pathways and interactions between multiple genes in gene therapy.     

Aromatic side-chain interactions in proteins. II. Near- and far-sequence Phe-X pairs

We have collected all aromatic pairs (3152) involving an N-phenyl partner in a dataset of 593 proteins of the PDB: 728 of these pairs involve a partner residue less than 6 apart in the sequence. These near-sequence Phe-X pairs correspond to specific conformations that stabilize secondary structures, mainly alpha-helices when the residues are 1, 3, and 4 apart, and beta-strands when they are 2 apart in the sequence. These conformations are not spatially random and have been examined in detail. The remaining phenylalanine pairs (2424) are between partners more than 5 apart in the sequence. Of these far-sequence pairs, 34% of occurrences are in sheets. Next in frequencies are pairs that bridge a beta-strand to a helix (24%), followed by pairs that bridge a beta-strand to a random coiled structure (15%). Helix to helix pairs only constitute 12% of these far-sequence pairs. Analysis of the pairing frequency supports the hypothesis that aromatic interactions are late events of protein folding.     

Aromatic side-chain interactions in proteins. I. Main structural features

In a data set of 593 nonhomologous proteins from the PDB, we have analyzed the pairing of phenylalanine, tyrosine, tryptophan, and histidine residues with their closest aromatic partner. The frequency distribution of the shortest interatomic distance of partners is bimodal with a sharp peak at approximately 3.8 A and a wider one at a longer distance. Only the 3.8 A peak corresponds to direct ring-ring interactions thus aromatic pairs. The aromatic pairs were separated into two classes, near-sequence pairs and far-sequence pairs. Near sequence pairs stabilize local structure, and far-sequence pairs stabilize tertiary structure. Far-sequence pairs (74% of all pairs) mainly bridge two beta-strands, followed by pairs that bridge a beta-strand and a helix, and pairs that bridge a beta-strand and a random coil structure. Pairs that bridge helices are rare. The secondary structure of the near-sequence pairs depends on the partner distance in the sequence. When the partners are 1, 3, or 4 residues apart in the sequence, pairs are mostly found in helical structures. When the partners are two apart, pairs are mostly found in the same beta-strand. Analysis of the frequency of near sequence pairs supports the hypothesis that aromatic pairing occurs after, rather than before, the formation of secondary structures.     

Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery

The function of the human nucleotide excision repair (NER) apparatus is to remove bulky adducts from damaged DNA. In an effort to gain insights into the molecular mechanisms involved in the recognition and excision of bulky lesions, we investigated a series of site specifically modified oligonucleotides containing single, well-defined polycyclic aromatic hydrocarbon (PAH) diol epoxide-adenine adducts. Covalent adducts derived from the bay region PAH, benzo[a]pyrene, are removed by human NER enzymes in vitro. In contrast, the stereochemically analogous N(6)-dA adducts derived from the topologically different fjord region PAH, benzo[c]phenanthrene, are resistant to repair. The evasion of DNA repair may play a role in the observed higher tumorigenicity of the fjord region PAH diol epoxides. We are elucidating the structural and thermodynamic features of these adducts that may underlie their marked distinction in biologic function, employing high-resolution nuclear magnetic resonance studies, measurements of thermal stabilities of the PAH diol epoxide-modified oligonucleotide duplexes, and molecular dynamics simulations with free energy calculations. Our combined findings suggest that differences in the thermodynamic properties and thermal stabilities are associated with differences in distortions to the DNA induced by the lesions. These structural effects correlate with the differential NER susceptibilities and stem from the intrinsically distinct shapes of the fjord and bay region PAH diol epoxide-N(6)-adenine adducts.     

The development of phenanthrene catabolism in soil amended with transformer oil

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants frequently associated with light non-aqueous-phase liquids (LNAPLs) in soil. Microbial degradation comprises a major loss process for PAHs in the environment. Various laboratory studies, using known degraders, have shown reduced or enhanced mineralisation of PAHs when dissolved in different LNAPLs. Effects due to the presence of LNAPLs on indigenous micro-organisms, however, are not fully understood. A pristine pasture soil was spiked with [14C]phenanthrene and transformer oil to 0, 0.01 and 0.1%, and incubated for 180 days. The catabolic potential of the soil towards phenanthrene was assessed periodically during ageing. The extent of the lag phase (prior to >5% mineralisation), maximum rates and overall extents of mineralisation observed during the course of a 14-day bioassay appeared to be dependent upon phenanthrene concentration, the presence of transformer oil, and soil-contaminant contact time. Putatively, transformer oil enhanced acclimation and facilitated the development of measurable catabolic activity towards phenanthrene in a previously uncontaminated pasture soil. Exact mechanisms for the observed enhancement, longer-term fate/degradation of the oil and residual phenanthrene, and effects of the presence of the oil on the indigenous microbes over extended time frames warrant further investigation.     

Screening of Pleurotus ostreatus isolates for their ligninolytic properties during cultivation on natural substrates

Thirteen basidiospore-derived isolates of Pleurotus ostreatus f6 strain differing in the level of ligninolytic enzyme production and other characteristics (mycelium extension rate, colony morphology) from the parental strain were cultivated on natural substrates. Under these conditions ligninolytic enzyme activity, loss of organic mass, polycyclic aromatic hydrocarbons (PAHs) degradation and colonization of sterile and nonsterile soil were studied. The activity of ligninolytic enzymes was substantially higher in straw than in liquid culture, although the differences between the isolates were less pronounced on this substrate. Some of the isolates showed a very good ability to decompose the lignocellulosic substrate (straw) and a relatively high loss of organic mass was found after 50 days of cultivation in these strains. The original strain f6 and isolates B13 and B26 successfully degraded all seven tested PAH compounds present in experimental soil samples, but the higher or lower ligninolytic enzyme production of isolates tested had no substantial effect on the extent of the degradation. In our screening, six basidiospore-derivedisolates growing well in nonsterile soil were found, whichcould be suitable for the prospective biotechnological exploitation.     

污染土壤中多环芳烃生物降解的调控研究

选用温度、湿度、表面活性剂ＴＷ８０和ＣＮＰ比４个因素为调控因子,采用正交法进行周期为１５０天的实验研究．结果表明,３０天后,土壤中ＰＡＨｓ的降解率可达４４．５～７４．６％,６０天后,达７０．４～９３．７％,降解率的不同与调控条件显著相关．在此期间,降解最佳条件为４０℃,湿度２５％,ＣＮＰ比为１２０１０１,ＴＷ８０分别为２００～５００ｍｇ·ｋｇ－１．实验结束时,土壤中ＰＡＨｓ的降解率达９１．２～９９．８％．降解的最佳条件是４０℃,湿度１５％．经Ｒ值判别表明,不同时期各因子对ＰＡＨｓ降解影响有所不同．温度对ＰＡＨｓ降解影响较大,表面活性剂对土壤中ＰＡＨｓ的生物降解有调控作用．