Studies on the Metabolism of Nitrogen Compounds by Prof J. W. Gorrod.

The scientific research papers by Professor John William Gorrod who died in December 2015 are reviewed. He was one of the most important scientists in the area and investigated mainly the biological oxidation of nitrogen containing drugs and xenobiotics. In this review, more than 200 papers belong to him are evaluated, most of which involved the results from in vitro microsomal oxidative metabolism of nitrogen containing xenobiotics. These studies include the carcinogenicity of tobacco smoke constituents, aromatic amines, aminophenols, rubber and aminobiphenyls, the assay for ferriheamoglobinemia forming compounds, metabolism of aralkyl azo compounds, aromatic amines, aromatic azaheterocycles, synthesis of N-oxidative metabolites i.e. N-oxides, nitrones and hydroxylamines, mutagenicity of amines, metabolism of adenine derivatives, alicyclic amines and aromatic heterocyclic amines, in vivo and in vitro metabolism of nicotine and related tobacco alkaloids, effects of various substrates on MAO enzyme and reviews related to nicotine and N-oxidation of other nitrogen containing compounds. In the major part of these studies, the spectroscopic and chromatographic methods were developed to evaluate and determine the investigated compounds.

Synthesis, biological evaluation, and computational studies of Tri- and tetracyclic nitrogen-bridgehead compounds as potent dual-acting AChE inhibitors and hH3 receptor antagonists.

Combination of AChE inhibiting and histamine H3 receptor antagonizing properties in a single molecule might show synergistic effects to improve cognitive deficits in Alzheimer's disease, since both pharmacological actions are able to enhance cholinergic neurotransmission in the cortex. However, whereas AChE inhibitors prevent hydrolysis of acetylcholine also peripherally, histamine H3 antagonists will raise acetylcholine levels mostly in the brain due to predominant occurrence of the receptor in the central nervous system. In this work, we designed and synthesized two novel classes of tri- and tetracyclic nitrogen-bridgehead compounds acting as dual AChE inhibitors and histamine H3 antagonists by combining the nitrogen-bridgehead moiety of novel AChE inhibitors with a second N-basic fragment based on the piperidinylpropoxy pharmacophore with different spacer lengths. Intensive structure-activity relationships (SARs) with regard to both biological targets led to compound 41 which showed balanced affinities as hAChE inhibitor with IC50 = 33.9 nM, and hH3R antagonism with Ki = 76.2 nM with greater than 200-fold selectivity over the other histamine receptor subtypes. Molecular docking studies were performed to explain the potent AChE inhibition of the target compounds and molecular dynamics studies to explain high affinity at the hH3R.

Neutrophil uptake of nitrogen-bisphosphonates leads to the suppression of human peripheral blood γδ T cells.

Nitrogen-bisphosphonates (n-BP), such as zoledronate, are the main class of drugs used for the prevention of osteoporotic fractures and the management of cancer-associated bone disease. However, long-term or high-dose use has been associated with certain adverse drug effects, such as osteonecrosis of the jaw and the loss of peripheral of blood Vγ9Vδ2 T cells, which appear to be linked to drug-induced immune dysfunction. In this report we show that neutrophils present in human peripheral blood readily take up zoledronate, and this phenomenon is associated with the potent immune suppression of human peripheral blood Vγ9Vδ2 T cells. Furthermore, we found this zoledronate-mediated inhibition by neutrophils could be overcome to fully reconstitute Vγ9Vδ2 T cell proliferation by concomitantly targeting neutrophil-derived hydrogen peroxide, serine proteases, and arginase I activity. These findings will enable the development of targeted strategies to mitigate some of the adverse effects of n-BP treatment on immune homeostasis and to improve the success of immunotherapy trials based on harnessing the anticancer potential of peripheral blood γδ T cells in the context of n-BP treatment.

Radiolabeled bombesin analogs for prostate cancer diagnosis: preclinical studies.

INTRODUCTION: Radionuclide imaging can be a useful tool for the diagnosis of prostate cancer. Bombesin (BBN) is a molecule with high affinity for gastrin releasing peptide (GRP) receptors which are over-expressed in that tumor. This report compares (99m)Tc-HYNIC-betaAla-BBN(7-14)NH2 [(99m)Tc-HYNIC-BBN] and (99m)Tc identical withN(PNP6)-Cys-betaAla-BBN(7-14)NH2 [(99m)TcN(PNP6)-Cys-BBN] with regard to labeling procedures as well as in vitro and in vivo evaluation (biodistribution and scintigraphic imaging). METHODS: Peptide synthesis was performed in an automated peptide synthesizer. HYNIC-BBN was radiolabeled with pertechnetate using tricine and ethylenediamine diacetic acid (EDDA) as coligands. Cys- BBN was radiolabeled in a two-step procedure with the preparation of the precursor (99m)Tc-Nitrido first and then introducing diphosphine (PNP6). Radiochemical evaluation of conjugates, as well as studies of stability, transchelation toward cysteine, and partition coefficient were done. Biological studies included internalization, biodistribution in healthy animals and in animals bearing PC3 cancer cells with acquisition of images from the tumor-bearing animals. RESULTS: Both complexes showed a high radiochemical yield along with good stability. Biodistribution studies pointed out strong renal excretion for the former complex due to its hydrophilic profile and marked hepatobiliary excretion for the latter, corresponding to observed lipophilicity. Tumor uptake was higher for (99m)Tc-HYNIC-BBN and the same occurred with internalization findings, which exceeded those of (99m)TcN(PNP6)-BBN. Blocking studies in mice bearing PC-3 tumor cells revealed significantly reduced pancreas and tumor uptake, demonstrating receptor specificity of the conjugates. CONCLUSION: The best radiotracer was (99m)Tc-HYNIC-BBN on the basis of high radiochemical yield, fast radiolabeling procedure without need for a purification step, and more consistent tumor uptake.

Evaluation of novel cationic (99m)Tc-nitrido complexes as radiopharmaceuticals for heart imaging: improving liver clearance with crown ether groups.

This report describes the evaluation of a series of novel cationic (99m)Tc-nitrido complexes, [(99m)TcN(DTC)(PNP)]+ (DTC = crown ether-containing dithiocarbamates; PNP = bisphosphine), as potential radiotracers for myocardial perfusion imaging. Synthesis of cationic (99m)Tc-nitrido complexes was accomplished in two steps according to literature methods. Biodistribution studies were performed in rats. Planar images of Sprague-Dawley rats administered with 15+/-2 MBq of cationic (99m)Tc radiotracer were obtained using a PhoGama large field-of-view Anger camera. Samples from both urine and feces were analyzed by a reversed-phase radio-HPLC method. Results from biodistribution studies showed that most of the cationic (99m)Tc-nitrido complexes have a high initial heart uptake with a long myocardial retention. They also show a rapid clearance from the liver and lungs. Cationic complexes [(99m)TcN(L2)(L6)]+ and [(99m)TcN(L4)(L6)]+ show heart/liver ratios four to five times better than that of (99m)Tc-sestamibi due to their much faster liver clearance. Their heart uptake and heart/liver ratio are comparable to that of (99m)TcN-DBODC5 within the experimental error. These findings have been confirmed by the results from imaging studies. Radio-HPLC analysis of urine and feces samples indicated that there was very little metabolism of cationic (99m)Tc-nitrido complexes in rats under anesthesia. The key finding of this study is that lipophilicity remains the most important factor affecting both heart uptake and target-to-background (T/B) ratios. Crown ethers are very useful functional groups to improve the liver clearance of cationic (99m)Tc-nitrido complexes. It is the combination of the appropriate DTCs and bisphosphines that results in cationic (99m)Tc-nitrido complexes with high heart uptake and fast clearance from the liver at the same time. The fast liver clearance of [(99m)TcN(L2)(L6)]+ and [(99m)TcN(L4)(L6)]+ suggests that they might be used to obtain clinically useful images as early as 30 min postinjection. [(99m)TcN(L2)(L6)]+ and [(99m)TcN(L4)(L6)]+ are very promising candidates for further evaluation in more extensive preclinical animal models.

Nitrogen dynamics under Lolium perenne after a single application of three different sewage sludge types from the same treatment stream.

Three sludge types from the same treatment stream (undigested liquid, anaerobically digested liquid and dewatered, anaerobically digested cake) were used in a field based tub study. Amendments (4, 8, and 16 Mg dry solid (ds)ha(-1)) were incorporated into the upper 15 cm of a sandy loam soil prior to sowing with rye-grass (Lolium perenne L.). Nitrogen transformations in the soil were determined for the 80 d period following incorporation. Nitrogen uptake and crop yield were measured in the cut sward 35 and 70 d after sowing. The study showed that application of sewage sludge at rates as low as 4 Mgha(-1) can have a nutritional benefit to rye-grass over the two harvests. Differences in N transformation, and hence crop nutritional benefit, between sludge types were evident throughout the experiment. In particular, the dewatering process changed the mineral N characteristics of the anaerobically digested sludge, which, when not dewatered, outperformed the other sludges in terms of yield and mineralisation rate at both harvests. The dewatered sludge produced the lowest yield of rye-grass. The undigested liquid sludge had the lowest foliar N and soil NO(3)-N concentrations, possibly immobilised as the large oxidisable C component of this sludge was metabolised by the microbial biomass. Correlation data support the concept of preferential uptake of NH(4)-N over NO(3)-N in Lolium perenne. Results are discussed in the context of managing sludge type and application for a plant nutrient source and NO(3)-N release.

Phycoremediation: key issues for cost-effective nutrient removal processes.

Phycoremediation applied to the removal of nutrients from animal wastewater and other high organic content wastewater is a field with a great potential and demand considering that surface and underground water bodies in several regions of the world are suffering of eutrophication. However, the development of more efficient nutrient removal algal systems requires further research in key areas. Algae growth rate controls directly and indirectly the nitrogen and phosphorus removal efficiency. Thus, maximum algae productivity is required for effective nutrient removal and must be considered as a key area of research. Likewise, low harvesting costs are also required for a cost-effective nutrient removal system. The use of filamentous microalgae with a high autoflocculation capacity and the use of immobilized cells have been investigated in this respect. Another key area of research is the use of algae strains with special attributes such as tolerance to extreme temperature, chemical composition with predominance of high added value products, a quick sedimentation behavior, or a capacity for growing mixotrophically. Finally, to combine most of the achievements from key areas and to design integrated recycling systems (IRS) should be an ultimate and rewarding goal.

Lung hyperpermeability and asthma prevalence in schoolchildren: unexpected associations with the attendance at indoor chlorinated swimming pools.

AIMS: To study whether exposure to nitrogen trichloride in indoor chlorinated pools may affect the respiratory epithelium of children and increase the risk of some lung diseases such as asthma. METHODS: In 226 healthy children, serum surfactant associated proteins A and B (SP-A and SP-B), 16 kDa Clara cell protein (CC16), and IgE were measured. Lung specific proteins were measured in the serum of 16 children and 13 adults before and after exposure to NCl(3) in an indoor chlorinated pool. Relations between pool attendance and asthma prevalence were studied in 1881 children. Asthma was screened with the exercise induced bronchoconstriction test (EIB). RESULTS: Pool attendance was the most consistent predictor of lung epithelium permeability. A positive dose-effect relation was found with cumulated pool attendance and serum SP-A and SP-B. Serum IgE was unrelated to pool attendance, but correlated positively with lung hyperpermeability as assessed by serum SP-B. Changes in serum levels of lung proteins were reproduced in children and adults attending an indoor pool. Serum SP-A and SP-B were already significantly increased after one hour on the pool side without swimming. Positive EIB and total asthma prevalence were significantly correlated with cumulated pool attendance indices. CONCLUSIONS: Regular attendance at chlorinated pools by young children is associated with an exposure dependent increase in lung epithelium permeability and increase in the risk of developing asthma, especially in association with other risk factors. We therefore postulate that the increasing exposure of children to chlorination products in indoor pools might be an important cause of the rising incidence of childhood asthma and allergic diseases in industrialised countries. Further epidemiological studies should be undertaken to test this hypothesis.

Preparation of new nitrogen-bridged heterocycles. 53. Syntheses of 3-(benzylthio)thieno[3,4-b]indolizine derivatives and their intramolecular arene-arene interactions.

Various ethyl 1-arylcarbonyl-3-[(un)substituted methylthio]thieno[3,4-b]indolizine-9-carboxylates were synthesized in good yields by a novel methodology in which the S-alkylation of 5-arylcarbonyl-4-ethoxycarbonylmethyl-3-(1-pyridinio)thiophene-2-thiolates with alkyl or benzyl halides, the 1,5-dipolar cyclization of the resulting pyridinium salts in the presence of a base, and the aromatization were performed. In the X-ray analyses of some 3-(benzylthio)thieno[3,4-b]indolizine-9-carboxylates, a gauche and two anti conformers in relation to the exocyclic sulfide linkage were found. Interestingly, all of the 3-(benzylthio)thieno[3,4-b]indolizine derivatives showed significant high-field shifts (delta up to 0.3 ppm) for the 5- and 6-proton signals compared with those of the 3-methylthio derivatives in the (1)H-NMR spectra and exhibited a definite absorption band near 425 nm in their UV spectra, indicating an intramolecular arene-arene interaction between the thieno[3,4-b]indolizine and the phenyl ring.

N-oxidation of drugs associated with idiosyncratic drug reactions.

Circumstantial evidence strongly suggests that most idiosyncratic drug reactions are due to reactive metabolites of drugs rather than due to the drugs themselves. Many of the drugs that are associated with idiosyncratic drug reactions contain nitrogen. There are many possible reasons for this association. One is simply that many drugs, especially CNS active drugs, contain nitrogen. In addition, nitrogen is relatively easy to oxidize because of its lone pair of electrons and many nitrogen-containing compounds readily undergo redox cycling, which can generate reactive oxygen species. There are several nitrogen-containing function groups that are especially associated with adverse reactions. These include aromatic amines, nitro compounds (nitro compounds are reduced to the same reactive intermediates as are formed by oxidation of the corresponding aromatic amine), hydrazines and compounds that can be oxidized to iminoquinone and related compounds. A greater attention to the issue of reactive metabolites during drug development would likely lead to safer drugs; however, not all drugs that form reactive metabolites are associated with a high incidence of idiosyncratic drug reactions. In addition to the presence of such a group, other factors, such as dose and electron density of the compound, appear to play a role in whether the drug containing such functional groups will be associated with a relatively high incidence of idiosyncratic drug reactions.

Influence of photoperiod on the uptake of nitrogen and phosphorus in the water by Eichhornia crassipes and Salvinia auriculata

The main goal of this research was to quantify the concentrations of total and ammonium N, nitrate, total and soluble P in the water in the presence of Eichhornia crassipes and Salvinia auriculata, which were submitted to two different photoperiods in a 24 hours incubation period in the laboratory. The macrophytes were incubated in plastic vials of approximately 1.5 litters, with a previously prepared solution with NH4NO3, NH4Cl, and KH2PO4. Eichhornia crassipes showed the highest average rate of reduction of all the nutrients analysed in relation to Salvinia auriculata. The largest photoperiod reflected in a higher average rate of reduction of nutrients, in both plants. Therefore, we may expect that in some periods of the year (e.g. summer), the aquatic macrophytes would show higher growth rates and higher rates of nitrogen and phosphorus absorption. These results are important for the implementation and management of wastewater treatment systems in tropical areas using aquatic macrophytes

[Nutrition of wheat under wet injury and its regulation].

With pot experiment, this paper dealt with the effects of different nutrition regulations on nutrient absorption and crop yield of wheat under waterlogged condition. N, P, K, Zn, Cu uptakes by wheat were greatly inhibited by waterlogging. The total amounts of reusable elements N, P, K and Zn in leaves decreased obviously in stems but did not in ears, compared with control; while the total amount of element Cu in all parts was reduced by waterlogging. The yield of individual wheat plant was significantly reduced under waterlogging. Additional N-fertilizer or spraying N, P and K significantly increased the uptake of nutrients and the yield for Wanmai 19 under light waterlogging, but were not effective for Yanmai 158 under heavy waterlogging. A positive correlation was found between the yield of wheat under waterlogging and the N accumulation in leaves.

[Biotransformation of nitrogen-containing compounds in the process of the continuous final purification of effluents from swine-breeding farms by immobilized cells]. / Biotransformatsiia azotsoderzhashchikh soedinenii v protsesse neprepyvnoi doochistki stokov svinokompleksov immobilizovannymi kletkami.

After their two-step purification in aeration tanks, effluents from a swine-breeding farm were subjected to final purification in a flow bioreactor with immobilized cells derived from activated sludge. The dynamics of nitrogen compounds were studied at the reactor inlet and outlet at three flow rates. The use of a simple mathematical model of the nitrogen balance allowed the rates of nitrogen assimilation-mineralization two nitrification stages, and nitrogen fixation-denitrification to be estimated. The first stage of nitrification was found to proceed intensely and produce high nitrite concentrations (up to 100 mg N/l, accounting for about half of the total N at the inlet). The second nitrification stage was inhibited, and nitrate was only produced after 3-4 weeks of continuous operation. The strategy of the final purification of effluents from nitrogen compounds is discussed on the basis of the known regulatory mechanisms of microbial activities in activated sludge.

Irradiation enhances cellular uptake of carboplatin.

PURPOSE: Because radiation is known to damage cellular membranes, the purpose of this study was to determine whether irradiation of cultured cells might modify the cellular uptake of the chemotherapy agent carboplatin. METHODS AND MATERIALS: Total intracellular platinum was measured using atomic absorption spectrometry in cultured V79 cells and in four Chinese hamster ovary (CHO) cell lines. RESULTS: Intracellular carboplatin concentrations increased linearly with radiation dose (10-50 Gy) under both hypoxic and oxic irradiation conditions. Similar doses of radiation did not significantly increase the uptake of a nontoxic platinum compound [Pt(NH3)4Cl2.H2O] (p > 0.5). Compared to unirradiated controls, there was no increase in intracellular carboplatin concentrations when carboplatin was irradiated prior to administration to the cell cultures (p > 0.5). Within the 32.5 min or less required to deliver the radiation, a dose of 50 Gy produced approximately a 50% increase in intracellular platinum in V79 cells and approximately an increase of a factor of 1.3-1.6 in the CHO cell lines. Although the increase in drug uptake would be expected to be less than 10% for most cell lines at the doses of radiation used to investigate radiosensitization by carboplatin, this level of increase may play a significant role in the radioenhancement observed in UV41 cells because these excision-repair--deficient cells are much more sensitive to carboplatin as measured by cytotoxicity. CONCLUSION: These results suggest that some of the enhanced cell killing that results when cells are exposed to carboplatin in combination with radiation may be attributed to an increased cellular uptake. One mechanism of radiopotentiation may be an enhanced chemotoxicity resulting from a radiation-induced increase in carboplatin uptake.

Nitrogen uptake by wheat seedlings, interactive effects of four nitrogen sources: NO3-, NO2-, NH4+, and urea.

The net influx (uptake) rates of NO3-, NH4+, NO2-, and urea into roots of wheat (Triticum aestivum cv Yecora Rojo) seedlings from complete nutrient solutions containing all four compounds were monitored simultaneously. Although urea uptake was too slow to monitor, its presence had major inhibitory effects on the uptake of each of the other compounds. Rates of NO3-, NH4+, and NO2- uptake depended in a complex fashion on the concentration of all four N compounds. Equations were developed which describe the uptake rates of each of the compounds, and of total N, as functions of concentrations of all N sources. Contour plots of the results show the interactions over the range of concentrations employed. The coefficients of these equations provide quantitative values for evaluating primary and interactive effects of each compound on N uptake.