Biopterin metabolism and phenylalanine hydroxylase activity during early liver regeneration

Rat liver biopterin content and the activities of two enzymes involved in biopterin metabolism, sepiapterin reductase and dihydropteridine reductase, were not altered twenty-four hours after partial hepatectomy. This surgical procedure did, however, produce a vigorous regenerative response as verified by an increase in ornithine decarboxylase activity. The tetrahydrobiopterin-dependent activity of phenylalanine hydroxylase was increased in homogenates of regenerating liver. The pteridine requirements for the expression of this activation, and the behavior of the enzyme on calcium-phosphate cellulose columns suggest that elevated levels of cyclic adenosine monophosphate in regenerating liver induce phosphorylation and activation of phenylalanine hydroxylase. This increase in the activity of the primary enzyme of phenylalanine catabolism was interpreted as a compensatory response designed to maintain homeostasis prior to liver regeneration.     

Release of mutagenic metabolites of benzo[a]pyrene from the perfused rat liver after inhibition of glucuronidation and sulfation by salicylamide

The role of glucuronide and sulfate conjugation in presystemic inactivation of benzo[a]pyrene (BP) metabolites was investigated with rat livers perfused with BP (12 mumol). Comparisons were made between metabolite profiles and mutagenicity of medium from perfusions with and without salicylamide, a selective inhibitor of glucuronide and sulfate conjugation. After 4 h perfusion in the presence of salicylamide, certain BP metabolites (diols, quinones, phenols, and metabolites more polar than BP-9,10-diol) were significantly increased at the expense of quinones and phenols in the glucuronide fraction. Mutagenicity of medium (detected by the Ames test, using tester strains TA98 and TA100) was low in perfusion without salicylamide. Mutagenicity detected with tester strain TA98 was significantly increased in perfusions with salicylamide. Involvement of glucuronidation in BP inactivation was also observed at the subcellular level; when cofactors of glucuronidation were added to liver homogenates along with the NADPH regenerating system in the Ames test, BP mutagenicity was markedly decreased. Both the activation of BP to mutagenic metabolites and the inactivation of BP metabolites by glucuronidation was much more pronounced with liver homogenates from 3-methylcholanthrene-treated rats than with those from phenobarbital-treated animals or untreated controls. The results suggest an important role for glucuronidation and sulfation in the inactivation and elimination of polycyclic aromatic hydrocarbons.     

The cyanobacterium Anabaena sp. PCC 7120 has two distinct beta-carotene ketolases: CrtO for echinenone and CrtW for ketomyxol synthesis

Two beta-carotene ketolases, CrtW and CrtO, are widely distributed in bacteria, although they show no significant sequence homology with each other. The cyanobacterium Anabaena sp. PCC 7120 was found to have two homologous genes. In the crtW deleted mutant, myxol 2'-fucoside was present, but ketomyxol 2'-fucoside was absent. In the crtO deleted mutant, beta-carotene was accumulated, and the amount of echinenone was decreased. Therefore, CrtW catalyzed myxol 2'-fucoside to ketomyxol 2'-fucoside, and CrtO catalyzed beta-carotene to echinenone. This cyanobacterium was the first species found to have both enzymes, which functioned in two distinct biosynthetic pathways.     

Variable product specificity of solanesyl pyrophosphate synthetase

The distribution of polyprenyl pyrophosphates synthesized by the action of solanesyl pyrophosphate synthetase from Micrococcus luteus is dramatically changed depending on the Mg⁺⁺ concentration. When the metal ion concentration is higher than 5 mM, octaprenyl and solanesyl (nonaprenyl) pyrophosphate are synthesized predominantly. On the other hand, when the metal ion level is lower than 0.5 mM, a variety of polyprenyl pyrophosphates ranging in carbon chain length from C₁₅ to C₄₀ are formed. Heptaprenyl pyrophosphate is the longest of the products formed at 0.1 mM of Mg⁺⁺.     

The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: implications for the catalytic mechanism

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin and non-heme iron-dependent enzyme that hydroxylates L-Phe to l-Tyr using molecular oxygen as additional substrate. A dysfunction of this enzyme leads to phenylketonuria (PKU). The conformation and distances to the catalytic iron of both L-Phe and the cofactor analogue L-erythro-7,8-dihydrobiopterin (BH2) simultaneously bound to recombinant human PAH have been estimated by (1)H NMR. The resulting bound conformers of both ligands have been fitted into the crystal structure of the catalytic domain by molecular docking. In the docked structure L-Phe binds to the enzyme through interactions with Arg270, Ser349 and Trp326. The mode of coordination of Glu330 to the iron moiety seems to determine the amino acid substrate specificity in PAH and in the homologous enzyme tyrosine hydroxylase. The pterin ring of BH2 pi-stacks with Phe254, and the N3 and the amine group at C2 hydrogen bond with the carboxylic group of Glu286. The ring also establishes specific contacts with His264 and Leu249. The distance between the O4 atom of BH2 and the iron (2.6(+/-0.3) A) is compatible with coordination, a finding that is important for the understanding of the mechanism of the enzyme. The hydroxyl groups in the side-chain at C6 hydrogen bond with the carbonyl group of Ala322 and the hydroxyl group of Ser251, an interaction that seems to have implications for the regulation of the enzyme by substrate and cofactor. Some frequent mutations causing PKU are located at residues involved in substrate and cofactor binding. The sites for hydroxylation, C4 in L-Phe and C4a in the pterin are located at a distance of 4.2 and 4.3 A from the iron moiety, respectively, and at 6.3 A from each other. These distances are adequate for the intercalation of iron-coordinated molecular oxygen, in agreement with a mechanistic role of the iron moiety both in the binding and activation of dioxygen and in the hydroxylation reaction.     

Metabolism and nucleic acid binding of 7-fluoro-2-acetamidofluorene in rats: oxidative defluorination and apparent dissociation from hepatocarcinogenesis of 8-(N-arylamide) guanine adducts on DNA

The significance in hepatocarcinogenesis of various arylamine/amide adducts with nucleic acid was investigated by the use of comparison studies on several different parameters. Female Fischer and Sprague-Dawley rats are comparably sensitive to hepatocarcinogenesis by 2-acetamidofluorene (AAF), while male rats are more sensitive. 7-Fluoro-AAF is more carcinogenic in Sprague-Dawley rats than is AAF, but is strikingly so toward the liver of the female rat. Based on these observations, binding of both compounds to liver nucleic acids was determined for male and female Fischer rats at 1 and 3 days after a single injection of carcinogen, and in female Sprague-Dawley rats from 1 to 28 days after a single injection. As shown by others, no 8-(N-2-fluorenylacetamido)guanine adduct could be found in RNA or DNA of female Sprague-Dawley rats treated with AAF (nor was the corresponding 7-fluoro derivative detectable). These adducts were present, however, in comparable amounts in both male and female Fischer rats. The binding of 7-fluoro-AAF derivatives was higher than that of AAF derivatives in female Sprague-Dawley rats. Feeding of either AAF or 7-fluoro-AAF to Sprague-Dawley rats for 4 weeks before a single injection of [3H]7-fluoro-AAF resulted in reduction of the 8-(N-2-(7-fluoro)fluorenylacetamido)guanine adduct in males to undetectable levels in DNA and to 10% of control level in RNA. Non-acetylated adducts were increased in males, but decreased in females by AAF prefeeding; 7-fluoro-AAF prefeeding resulted in little change in adduct formation in females and in a major increase in non-acetylated adducts in males. AAF adducts disappeared from DNA more rapidly than did 7-fluoro-AAF adducts. Assay of the urinary metabolites from the animals in the prefeeding experiment showed that all compounds fed (including the non-hepatocarcinogens 4-acetamidobiphenyl and 2-acetamidophenanthrene) increased the proportion of N-hydroxy-7-fluoro-AAF among the metabolites. Defluorination of 7-fluoro-AAF to 7-hydroxy-AAF was also demonstrated and the ratio of 7-hydroxy-AAF to 5-hydroxy-7-fluoro-AAF was comparable to that observed for 7-hydroxy-AAF/5-hydroxy-AAF and AAF itself, suggesting that fluoro substitution does not increase activity by preventing detoxication.     

Influence of cell crowding on toxicity of nitroheterocycles

Cell density (no. of cells per unit area or volume) during drug treatment may play a role of considerable importance in the interpretation of drug toxicity experiments performed in vitro. Chinese hamster V-79 and mouse L-929 cells exposed to nitroheterocycles under aerobic conditions are considerably more sensitive to the cytotoxic effects of these drugs when incubated at low cell density (10² cells/cm² or 10⁴ cells/ml) than at higher cell density (10⁴ cells/cm² or 10⁶ cells/ml). This may be related to diffusion limitations when cells are in contact and to the ability of dense cell suspensions to inactivate drugs. In contrast, under anaerobic conditions, more toxicity is observed at high cell density than at low cell density, perhaps due to local effects of toxic metabolites. Toxicity appears to correlate with intracellular drug levels under both aerobic and hypoxic conditions.The chemical nature of the fluorescent species has not yet been determined. However, it is likely that loss of the nitro group reduces but does not abolish fluorescence. When L-cells were used to metabolize AF-2 under hypoxia so that only 20% of the parent compound remained (with nitro group intact), 50% of the fluorescence was still present (unpublished results). Cells incubated with AF-2 under air or nitrogen show little decrease in fluorescence intensity for several hours after drug removal suggesting that the fluorescent compound being observed was bound intracellularly. As yet, we have no reason to suspect that the fluorescent products bound under hypoxia differ from those bound under air. Therefore, cell density dependent toxicity of nitroheterocycles under aerobic conditions may be related to diffusion limitations when cells are in close contact, as well as drug inactivation by cells at high cell densities.     

Bacteriophage P22 development is temperature sensitive in thiolutin resistant mutants of Salmonella typhimurium.

Thiolutin resistant mutants of $\text{Salmonella}\text{typhimurium}$ can not support the development of phage P22 at high temperature (40°C). Although normal amounts of phage DNA and lysozyme are synthesised, very few infectious particles are formed at higher temperature. The results indicate the involvement of host function in phage development.     

Radioactive assay for aryl hydrocarbon hydroxylase. Improved method and biological importance

By addition of two volumes of a 1M aqueous KOH/dimethylsulfoxide ($\text{15}\text{85}$; v/v) mixture to the enzymatic incubation medium, it is possible to selectively extract the unmetabolized benzo(a)pyrene in hexane. Therefore, the radio-activity remaining in the water phase corresponds to all the in vitro synthesized metabolites. This isotopic method is very sensitive (2 × 10^?11 moles) and is almost insensitive to the room lighting. The aryl hydrocarbon hydroxylase activities found with this method are 2,3 and 10 times higher in the liver, lung and kidney respectively compared to those obtained with the fluorimetric method.     

Cellular and chemical reduction products of misonidazole

Misonidazole is readily reduced by zinc dust in aqueous solution in the presence of ammonium chloride. High pressure liquid chromatographic (HPLC) separation of the reduction mixture revealed the presence of three products. These were identified as the hydroxylamine, amine and the hydrazo derivative of misonidazole. There is evidence that the azoxy derivative was an intermediate in the reduction process. When the reduction was carried out in dilute solution (0.1 mg/ml), the hydroxylamine was the only product. In concentrated solution (20 mg/ml), the hydrazo derivative was the major product. When misonidazole was reduced with hydrogen using palladium as catalyst, the amine was the only detectable product. Of the three products, only the hydroxylamine was found to bind covalently to bovine albumin. In Chinese hamster ovary (CHO) cells under hypoxic conditions the amine was confirmed as one of the metabolites. There was no evidence for the presence of detectable amounts of the hydroxylamine in the cell extracts. These studies suggest that the hydroxylamine is probably the reactive reduction metabolite responsible for the in vivo and in vitro binding of misonidazole to cellular macromolecules.     

Isolation and characterization of a novel banana rhizosphere bacterium as fungal antagonist and microbial adjuvant in micropropagation of banana

AIM: Isolation and characterization of a bacterial isolate (strain FP10) from banana rhizosphere with innate potential as fungal antagonist and microbial adjuvant in micropropagation of banana. METHODS AND RESULTS: Bacterium FP10 was isolated from the banana rhizosphere and identified as Pseudomonas aeruginosa based on phenotypic, biochemical traits and sequence homology of partial 622-bp fragment of 16S ribosomal DNA (rDNA) amplicon, with the ribosomal database sequences. Strain FP10 displayed antibiosis towards fungi causing wilt and root necrosis diseases of banana. Production of plant growth hormone, indole-3-acetic acid (IAA), siderophores and phosphate-solubilizing enzyme in FP10 was determined. Strain FP10 tested negative for hydrogen cyanide, cellulase and pectinase, the deleterious traits for plant growth. Screening of antibiotic genes was carried out by polymerase chain reaction using gene-specific primers. Amplification of a 745-bp DNA fragment confirmed the presence of phlD, which is a key gene involved in the biosynthesis of 2,4-diacetylphloroglucinol (DAPG) in FP10. The antibiotic produced by FP10 was confirmed as DAPG using thin layer chromatography, high performance liquid chromatography and Fourier transform infrared and tested for fungal antibiosis towards banana pathogens. Procedures for encapsulation of banana shoot tips with FP10 are described. CONCLUSIONS: Strain FP10 exhibited broad-spectrum antibiosis towards banana fungi causing wilt and root necrosis. DAPG by FP10 induced bulb formation and lysis of fungal mycelia. Encapsulation of banana shoot tips with FP10 induced higher frequency of germination (plantlet development) than nontreated controls on Murashige and Skoog basal medium. Treatment of banana plants with FP10 enhanced plant height and reduced the vascular discolouration as a result of Fusarium oxysporum f. sp. cubense FOC. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the innate potential of fungal antibiosis by DAPG antibiotic and production of siderophore, plant-growth-promoting IAA and phosphatase, the strain FP10 can be used as biofertilizer as well as a biocontrol agent.     

The metabolism of a series of polycyclic hydrocarbons by mouse skin maintained in short-term organ culture

Investigations on the metabolism of ³H-labelled chrysene, benz[a]anthracene, 7-methylbenz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, benzo[a]pyrene, dibenz[a,c]anthracene and dibenz[a,h]anthracene by mouse skin maintained in short-term organ culture were carried out. Estimations of the distribution of the metabolites of each hydrocarbon present after 24 h showed that there were wide variations both in the rates at which the hydrocarbons were metabolised and in the amounts of metabolites covalently bound to skin macromolecules. All the hydrocarbons were metabolised to dihydrodiols, which were identified by comparison on high pressure liquid chromatography (HPLC) with the authentic compounds, and these were the same diols as those that were formed in previous experiments with rat-liver microsomal fractions. However, free dihydrodiols represented only relatively small proportions of the total amounts of metabolites formed. All the hydrocarbons yielded dihydrodiols of the type that could give rise to bay-region diol-epoxides, when further metabolised, some of which are thought to be involved in hydrocarbon carcinogenesis.     

Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine

We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

Structured summary of protein interactions

dicPAH and dicPAHbind by molecular sieving (View Interaction: 1, 2, 3, 4)     

Photooxidation products of 7,12-dimethylbenz[a]anthracene.

The principal products of the photooxidation of 7,12-dimethylbenz[a]-anthracene (DMBA) in aqueous solutions by photooxidation induced by laboratory lighting have been characterized by high performance liquid chromatograms (HPLC), ultraviolet and mass spectrograms and by comparisons with authentic samples. The products identified were the 7,12-epidioxy-7,12-dihydro-7-12-dimethyl-, 7,12-dione, 7-hydroxymethyl-12-methyl-, 12-hydroxymethyl-7-methyl-, 7-formyl-12-methyl-, 12-formyl-7-methyl-, and 12-hydroxy-12-methyl-7-one derivatives of benz[a]-anthracene. The HPLC profile of products is similar to that obtained from oxidation of DMBA by 'one-electron' reagents, singlet oxygen, or liver microsomal metabolism. The first points of attack are the 7- and 12- positions. The mechanism of photooxidation appears to be generation of singlet oxygen by photodynamic effect of DMBA. None of the products is photosensitizing, however.     

Binding of benzo[a]pyrene at the 1,3,6 positions to nucleic acids in vivo on mouse skin and in vitro with rat liver microsomes and nuclei

Loss of tritium from specific positions in [³H,¹⁴C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-³H,¹⁴C]benzo[a]pyrene(B[a]P), [1,3-³H,¹⁴C]B[a]P, [1,3,6-³H,¹⁴C]B[a]P, [6,7-³H,¹⁴C]B[a]P, and [7-³H,¹⁴C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from $\text{[}{}^3\text{H}\text{,}{}^{14}\text{C}\text{]}\text{B}\text{[a]}\text{P}$ was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.     

Effect of various substituents in the 6-position on the relative carcinogenic activity of a series of benzo[a]pyrene derivatives

To test the hypothesis that polycyclic aromatic hydrocarbons capable of being converted to a reactive ester of the mesohydroxymethyl metabolite would be carcinogenic, a series of 6-substituted derivatives of benzo[a]pyrene (B[a]P) were tested for carcinogenicity in Sprague-Dawley rats by subcutaneous injection of the compound in sesame oil on alternate days for 30 doses. At the 0.2-μmol dose level B[a]P, 6-acetoxymethyl(6-AcOCH₂)B[a]P, 6-hydroxymethyl(6-HOCH₂)B[a]P, 6-methyl(6-CH₃)B[a]P and 6-benzoyloxymethyl(6-BzOCH₂)B[a]P were nearly equipotent, 6-formyl(6-OCH)-and 6-chloromethyl(6-ClCH₂)B[a]P were less active, and 6-methoxymethyl (6-MeOCH₂)B[a]P was inactive. At lower doses the order of potency was estimated to be: 6-AcOCH₂- = 6-HOCH₂- = or > B[a]P > 6-CH₂- > 6-BzOCH₂- > 6-ClCH₂- > 6-OCH- > 6-BrCH₂B[a]P. Incubation of these compounds in the presence of cofactors or cofactors plus a microsomal preparation of rat subcutis indicated that enzymic activation was necessary for metabolism to highly polar products and for conversion of 6-AcOCH₂-, 6-BzOCH₂- and 6-OCHB[a]P to 6-HOCH₂B[a]P. The halomethyl compounds were converted to 6-HOCH₂B[a]P in the absence of enzyme by hydrolysis. 6-MeOCH₂B[a]P was unchanged in this system. These observations are consistent with the foregoing hypothesis with regard to derivatives of B[a]P and demonstrate that compounds of this series that are capable of conversion to the 6-HOCH₂-derivatives are carcinogenic.     

Metabolic transformation of 2-methylellipticinium

2-methyellipticinium (NSC 226137) does not exhibit any spectral interaction with cytochrome p-450; however, it is transformed $\text{in vitro}$ by microsomes from livers of phenobarbital induced rats. This transformation is NADPH dependant. According to presently available analytical criteria (HPLC and chromatographic behavior, UV and mass spectra), its product is very likely 2-methyl-9-hydroxyellipticinium (NSC 264137) which is an active antitumor drug in man. Two minor metabolites are also present. The same major product is found in the bile of non-induced rats after intravenous administration of 2-methylellipticinium.