Colin Charnock

Assimilable organic carbon and biodegradable dissolved organic carbon in Norwegian raw and drinking waters

Abstract Assimilable organic carbon (AOC) and biodegradable dissolved organic carbon (BDOC) were measured in Norwegian raw and drinking waters. Several of the water-works investigated produce a drinking water which is, or approximates to, biological stability according to the AOC analysis. The reduction of AOC by water treatment was shown to be a function of the raw water quality and the particular treatment process. Levels of AOC were generally low in Norwegian raw and drinking waters, and thus the situation appears promising with respect to controlling microbial aftergrowth during distribution. Coagulation-filtration (mainly direct filtration) trains and anionic exchange achieved significant reductions in AOC, whereas membrane filtration had little effect. Treatment plants using limited or no specific measures for removal of dissolved organic carbon (DOC) and postchlorination, increased AOC levels. Disinfection with UV light may produce less AOC than chlorination. BDOC was effectively reduced by all water treatments. Membrane filtration performed at least as well as other treatments in removing BDOC. This suggests that bulk BDOC is typically of higher molecular weight than AOC. Correlations between AOC and BDOC in raw and drinking waters were not significant. This finding supports the contention that these parameters are independent measures of the labile organic fraction of natural waters. It seems likely that the AOC and BDOC analyses target different fractions of the biodegradable organic material (BOM). When AOC and BDOC were used to evaluate the effectiveness of treatment processes the results were more promising. Correlation factors showed that these parameters generally provided complementary information about the fate of BOM during water treatment. However, because BDOC decreased during water treatment at all but one plant, whereas AOC often increased, necessitates that the raw data and the correlation factors be presented together in studies of this nature. The addition of nutrient salts in the AOC assay usually gave moderate increases in the values obtained. Consequently, nutrients other than organic carbon may limit aftergrowth in Norwegian drinking water. Use of an alternative bioassay organism gave lower AOC values, thus confirming the suitability of the standard assay organism.

Combining esters of para-hydroxy benzoic acid (parabens) to achieve increased antimicrobial activity

Background and objective:  Lower alkyl esters of p‐hydroxy benzoic acid (parabens) are widely used as preservatives of pharmaceuticals, cosmetics and allied products. Microbial activity increases with increasing alkyl chain length for the commonly used methyl, ethyl, propyl and butyl parabens, and synergy between parabens has been reported. However, although generally giving lower MIC values, greater toxicity issues seem to be associated with the larger parabens. Each of these observations is important in deciding how parabens should be combined to achieve best protection of the product in question, whilst minimizing the health risk to the user.

Are multidose over-the-counter artificial tears adequately preserved?

Purpose: To investigate artificial tears containing different preservatives for antimicrobial efficacy. Based on the challenge test outlined in the European Pharmacopoeia, products were tested in their original containers to see whether their component preservative had sufficient activity. Methods: Five brands of over-the-counter artificial tears each containing different preservatives (benzalkonium chloride/EDTA, parabens, chlorobutanol, silver chloride complex, and Purite-stabilized oxychloro complex) were inoculated with test microorganisms (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans). Changes in the microbial start concentration with time were followed by plating onto growth media to provide a measure of the preservative efficacy. In another test, artificial tears were applied to paper disks that were then placed onto agar growth media seeded with microorganisms. Zones of inhibition were measured after incubation. Results: Only the brand of artificial tears containing benzalkonium chloride/EDTA satisfied the major criteria for antimicrobial preservation for all the test microorganisms. Only a benzalkonium chloride/EDTA-containing disk placed on agar seeded with S. aureus produced a zone of inhibition in the agar diffusion test. Conclusion: The brand of artificial tears containing benzalkonium chloride/EDTA is suitable for sale in countries adopting the monographs of the European Pharmacopoeia. Other brands would only be suitable for sale if justified reasons for not meeting the major criteria for preservative efficacy can be provided.

Synthesis and antimycobacterial activities of non-purine analogs of 6-aryl-9-benzylpurines: Imidazopyridines, pyrrolopyridines, benzimidazoles, and indoles

6,9-Disubstituted purines and 7-deazapurines are known to be powerful inhibitors of Mycobacterium tuberculosis (Mtb) in vitro. Analogs modified in the six-membered ring (imidazopyridines, pyrrolopyridines, benzimidazoles, and indoles) were synthesized and evaluated as Mtb inhibitors. The targets were prepared by functionalization on the bicyclic heterocycle or from simple pyridines. The results reported herein, indicate that the purine N-1, but not N-3, is important for binding to the unknown target. The 3-deazapurines appears to be slightly more active compared to the parent purines and slightly less active than their 7-deazapurine isomers. Removal of both the purine N-3 and N-7 did not result in further enhanced antimycobacterial activity but the toxicity towards mammalian cells was increased. Both 3-deaza and 3,7-dideazapurines exhibited a modest activity against of the Mtb isolate in the state of non-replicating persistence.

Synthesis of non-purine analogs of 6-aryl-9-benzylpurines, and their antimycobacterial activities. Compounds modified in the imidazole ring.

Purine analogs modified in the five-membered ring have been synthesized and examined for antibacterial activity against Mycobacterium tuberculosis H(37)Rv in vitro employing the microplate alamar blue assay (MABA). The 9-deaza analogs were only found to be weak inhibitors, but the 8-aza-, 7-deaza- and 8-aza-7-deazapurine analogs studied displayed excellent antimycobacterial activities, some even substantially better than the parent purine. In the 7-deazapurine series, MIC values between 0.08 and 0.35 μM, values comparable or better than the reference drugs used in the study (MIC rifampicin 0.09 μM, MIC isoniazid 0.28 μM and MIC PA-824 0.44 μM). The five most active compounds were also examined against a panel of drug-resistant Mtb strain, and they all retained their activity. The compounds examined were significantly less active against M. tuberculosis in a state of non-replicating persistence (NRP). MIC in the low-oxygen-recovery assay (LORA) ≥ 60 μM. The 7-deazapurines were somewhat more toxic towards mammalian cells, but still the selectivity indexes were excellent. The non-purine analogs exhibit a selective antimycobacterial activity. They were essentially inactive against Staphylococcus aureus and Escherichia coli.

Identification and phylogeny of the small eukaryote population of raw and drinking waters

Culture-dependent and -independent methods were used to investigate the small eukaryote composition of raw and finished waters in the Norwegian cities of Oslo, Tromsø, Fredrikstad and Oppegård. Probes with general applicability to the 18S rRNA genes of the small eukaryote consortium were used for PCR-denaturing gradient gel electrophoresis (DGGE), and in the generation of clone libraries using the TOPO™ cloning and sequencing system. The chosen probes invariably gave a single band in agarose gel electrophoresis, indicating amplification of an area of similar size. DGGE and cloning analyses resolved the bands into components representing many unique amplicons. Diversity and composition in the collection were studied by DNA-sequencing, and visual examination of DGGE patterns. The cloning approach enabled the putative identification of a total of approximately 100 unique small eukaryotes. The major fraction of these represented ciliated and flagellated protozoal species. This was in keeping with the findings from protozoal cultivation. DNA from a number of multicellular eukaryotes was also detected. Amoebal and fungal DNA was rarely found. The latter may indicate a low incidence or a bias in the analysis technique. The population of small eukaryotes appears typical for pristine waters and no primary pathogens were detected by culture-independent techniques. However, the potentially pathogenic protozoa Acanthamoeba castellanii was grown on one occasion from Oslos drinking water. DGGE allowed the identification of fewer amplicons (by excision and sequencing of bands) than by the cloning-transformation approach. The DGGE analysis revealed clear similarities between the compositions of the raw and treated waters, indicating that cells or DNA in the raw water pass through the treatment trains. Protozoal culture and heterotrophic plate count analysis consistently revealed viable cells in both raw and treated waters in Oslo. This indicates that a fraction of the clone library represents eukaryotic species surviving the treatment trains. The analyses here presented represent the first published study of the general small eukaryotic fraction of the Capitals drinking water, and those of three other Norwegian cities. We suggest that DGGE profiles may have a value in judging physical treatment efficacy (removal of cells), but that direct cloning and sequencing studies is more amenable for characterization of uncultured microbes.

Synthesis and antimycobacterial activity of 5-formylaminopyrimidines; analogs of antibacterial purines.

Pyrimidine analogs of antimycobacterial purines have been synthesized and their biological activities evaluated. Several 5-formamidopyrimidines exhibited profound activity against Mycobacterium tuberculosis in vitro (IC(90)< or =1.5 microg/mL), and they were essentially inactive against other bacteria.

Antimicrobial and Antineoplastic Activities of Agelasine Analogs Modified in the Purine 2-Position

Agelasines are 7,9‐dialkylpurinium salts found in marine sponges (Agelas sp.), which display a variety of antimicrobial and cytotoxic effects. We have synthesized simplified agelasine analogs modified in the purine 2‐position and examined their antimicrobial and anticancer activities. The compounds were screened against Staphylococcus aureus, Escherichia coli, Mycobacterium tuberculosis, Candida krusei, and Candida albicans, protozoa causing tropical diseases (Plasmodium falciparum, Leishmania infantum, Trypanosoma cruzi, and Trypanosoma brucei), a panel of human cancer cell lines (U‐937 GTB, RPMI 8226/s, CEM/s, and ACHN) as well as VERO and/or MRC‐5 cells. The results indicate that the introduction of a methyl group in the purine 2‐position is beneficial for antimycobacterial and antiprotozoal activity, and that amino groups may enhance activity against several cancer cell lines.

Activity of 6-aryl-pyrrolo[2,3-d]pyrimidine-4-amines to Tetrahymena

A series 6-aryl-pyrrolo[2,3-d]pyrimidine-4-amines (43 compounds), some of which are epidermal growth factor tyrosine kinase inhibitors, were tested for their protozoal toxicity using an environmental Tetrahymena strain as model organism. The protozoacidal activity of the analogues was found to be highly dependent on a 4-hydroxyl group at the 6-aryl ring, and a chiral 1-phenylethanamine substituent in position 4. Further, the potency was affected by the aromatic substitution pattern of the phenylethanamine: the unsubstituted, the meta-fluoro and the para-bromo substituted derivatives had the lowest minimum protozoacidal concentrations (8-16 μg/mL). Surprisingly, both enantiomers were found to have high potency suggesting that this compound class could have several modes of action. No correlation was found between the compounds protozoacidal activity and the in vitro epidermal growth factor receptor tyrosine kinase inhibitory potency. This suggests that the observed antimicrobial effects are related to other targets. Testing towards a panel of kinases indicated several alternative modes of action.

2-Substituted agelasine analogs : synthesis and biological activity, and structure and reactivity of synthetic intermediates

2-Substituted N-methoxy-9-methyl-9H-purin-6-amines were synthesized either from their corresponding 6-chloro-9-methyl-9H-purines or 2-chloro-N-methoxy-9-methyl-9H-purin-6-amine. Great diversity in the amino/imino tautomeric ratios was observed and calculated based on 1H NMR. The tautomers were identified by 1D and 2D 1H, 13C, and 15N NMR techniques, and showed significant variation both in 13C and 15N shift values. Comparison of the tautomeric ratios with Hammett F values revealed that as the field/inductive withdrawing abilities of the 2-substituent increased, the ratio of amino:imino tautomers was shifted toward the amino tautomer. Computational chemistry exposed the significance of hydrogen bonding between solvent and the compound in question to reach accurate predictions for tautomeric ratios. B3LYP/def2-TZVP density functional theory (DFT) calculations resulted in quantitatively more accurate predictions than when employing the less expensive BP86 functional. N-7-Alkylation of the 2-substituted N-methoxy-9-methyl-9H-purin-6-amines showed that when the field/inductive withdrawing ability of the 2-substituent reached a certain point the reactivity drastically dropped. This correlated with the atomic charges on N-7 calculated using a natural bond orbital (NBO) analysis. Biological screening of the final 2-substituted agelasine analogs indicated that the introduction of a methyl group in the 2-position is advantageous for antimycobacterial and antiprotozoal activity, and that an amino function may improve activity against several cancer cell lines.