The effects of folate intake on DNA and single-carbon pathway metabolism in the fruit fly Drosophila melanogaster compared to mammals.

Mechanisms of vitamin function in non-mammals are poorly understood, despite being essential for development. Folate and cobalamin are B-vitamin cofactors with overlapping roles in transferring various single-carbon units. In mammals, one or both is needed for nucleotide synthesis, DNA methylation, amino acid conversions and other reactions. However, there has been little investigation of the response to folate or cobalamin in insects. Here, we manipulated folate intake and potentially cobalamin levels in the fruit fly Drosophila melanogaster with chemically-defined diets, an antibiotic to reduce bacterially-derived vitamins, and the folate-interfering pharmaceutical methotrexate, to see if single-carbon metabolites and DNA synthesis rates would be affected. We found that similar to mammals with low folate intake, fruit fly larvae had significantly slower growth and DNA synthesis rates. But changes to single carbon-metabolites did not mirror that of mammals with abnormal folate or given MTX. Five of the nine metabolites measured were not significantly affected (methionine, serine, glycine, methylglycine, and dimethylglycine) and three (cystathionine, methylgycine, and methylmalonic acid) were only decreased in larvae consuming methotrexate. Metabolites expected to be elevated if flies used cobalamin from microbial symbionts were not affected by dietary sulfaquinoxaline. Our data support the role of folate in nucleotide synthesis in D. melanogaster and that microbial symbionts provide functioning folates. We could not confirm how folate intake affects single carbon pathway metabolites, nor whether Drososphila use microbially-derived cobalamin. Further work should explore which cofactors are used in fruit flies in these important and potentially novel pathways.

Influence of three coccidiostats on the pharmacokinetics of florfenicol in rabbits.

In-feed Medication has been used for a long time to prevent coccidiosis, a worldwide protozoal disease in rabbits. Florfenicol (FFC) has been widely used in veterinary clinics for bacterial diseases treatment. Therefore, the use of combinations of coccidiostats with FFC in rabbits is common. In the present study, we aimed to evaluate the effect of three coccidiostats, sulfaquinoxaline (SUL), robenidine (ROB), and toltrazuril (TOL), as feed additives on the pharmacokinetic profile of FFC in rabbits. The disposition kinetics of FFC in rabbits were investigated after a single intravenous injection (25 mg/kg) in rabbits fed anticoccidial-free diets or feeds containing SUL (250 ppm), ROB (66 ppm), or TOL (2 ppm), respectively, for 20 days. Plasma FFC concentrations were determined by the high performance liquid chromatography (HPLC) method. The pharmacokinetic parameters of FFC were analyzed using a non-compartmental analysis based on the statistical moment theory. The results demonstrated that ROB feeding resulted in an obvious decrease in plasma FFC level as compared with anticoccidial-free feeding. The terminal elimination half-life (t1/2z), area under the concentration-time curve (AUC), area under the first moment curve (AUMC), and mean residence time (MRT) significantly decreased, whereas the elimination rate constant (λz) and total body clearance (CLz) obviously increased in rabbits pretreated with ROB. However, we did not find that SUL or TOL feeding had any effect on the pharmacokinetic profile of FFC. Our findings suggested that more attention should be paid to the use of FFC in rabbits supplemented with ROB.

Sulfa drugs as inhibitors of carbonic anhydrase: new targets for the old drugs.

Sulfa drugs are well-known antibacterial agents containing N-substituted sulfonamide group on para position of aniline ring (NH2RSO2NHR'). In this study 2,4-dichloro-1,3,5-triazine derivatives of sulfa drugs, sulfamerazine (1b), sulfaquinoxaline (2b), sulfadiazine (3b), sulfadimidine (4b), and sulfachloropyrazine (5b) (1a-5a) were synthesized and characterized. Their carbonic anhydrase inhibition activity was evaluated against bovine cytosolic carbonic anhydrase isozyme II (bCA II). For the sake of comparison the CA inhibition activity of the parent sulfa drugs (1b-5b) was also evaluated. A significant increase in CA inhibition activity of sulfa drugs was observed upon substitution with 2,4-dichloro-1,3,5-triazine moiety. Molecular docking studies were carried out to highlight binding site interactions. ADME properties were calculated to evaluate drug likeness of the compounds.

Immunosuppressive properties of chloroquinoxaline sulfonamide.

Chloroquinoxaline sulfonamide (CQS), a sulfanilamide derivative with antitumor activity, was found to be toxic to lymphoid tissue during preclinical studies. The mechanism of this toxicity appears to involve profound inhibition of lymphocyte activation. Incubation of human peripheral blood mononuclear cells (PBMNCs) with CQS decreased cellular incorporation of thymidine and deoxyuridine in a dose-dependent manner. Analysis of cell cycle distribution by flow cytometry indicated that CQS blocked movement out of the G0/G1 phase. Drug-treated cells were smaller and expressed fewer receptors for interleukin-2 (IL-2) and transferrin than untreated mitogen-stimulated lymphocytes. These observations support the notion that CQS has cell cycle specificity in regulating lymphocyte proliferation. As little as 10 microM CQS markedly inhibited both human lymphocyte and murine CTLL cell replication in response to IL-2 containing growth factors. However, CQS did not block secretion of IL-2 into culture supernatant fractions by human PBMNCs. Finally, CQS inhibited in vitro production of immunoglobulins G and M by mitogen-stimulated lymphocytes, primarily by causing cytotoxicity. In all of these drug effects, CQS was approximately one to two logs more potent than the parent compound, sulfaquinoxaline (SQ). These studies indicate that CQS inhibits essential basic processes in human lymphocytes. This agent may find use as an immunosuppressive drug.

Sulfaquinoxaline inhibition of vitamin K epoxide and quinone reductase.

Sulfaquinoxaline (N1-(2-quinoxalinyl)sulfanilamide) has been shown to be a potent (Ki = 1 microM) freely reversible inhibitor of the dithiothreitol-dependent reduction of both vitamin K epoxide and vitamin K quinone by rat liver microsomes in vitro. This observation provides an explanation for the hemorrhagic syndrome occasionally seen in poultry on medicated feed and the efficacy of sulfaquinoxaline in anticoagulant based rodenticides. Sulfaquinoxaline inhibition resembled inhibition by coumarin anticoagulants (e.g., warfarin) and hydroxynaphthoquinones (e.g., lapachol). Inhibition was observed in assays using microsomes from control strain rats, but the enzyme was resistant to sulfaquinoxaline in microsomes from warfarin-resistant rats. Steady-state kinetics inhibition patterns were nearly competitive versus dithiothreitol and nearly uncompetitive versus vitamin K epoxide as is observed for warfarin and lapachol. These results suggest that this inhibitor binds to the oxidized form of vitamin K epoxide reductase in the same way as suggested for the coumarins and hydroxyquinones. Of 10 other sulfa drugs tested, none were inhibitors, and of fragments and related compounds tested, only 2-aminoquinoxaline benzenesulfonamide was active. These results provide a probably orientation in the binding site in relation to that for warfarin and lapachol.

Cellular pharmacology of chloroquinoxaline sulfonamide and a related compound in murine B16 melanoma cells.

Chloroquinoxaline sulfonamide (CQS), a chlorinated derivative of sulfaquinoxaline (SQ), inhibited proliferation of murine B16 melanoma cells, but only when relatively high drug concentrations (1 mM) were used. The inhibition of cell growth by CQS was at least partially reversible by incubation in drug-free medium. Incubation of melanoma cells with CQS was associated with an arrest of the cell cycle in G0/G1 as measured by flow cytometry. The drug slightly decreased uptake of radiolabeled deoxyuridine and thymidine after 24- and 48-hr incubation periods but increased nucleoside incorporation at 72 hr. No evidence of intercalation with DNA was found. Because SQ previously was reported to inhibit an aspect of folate metabolism, we investigated the possibility that CQS limits tumor cell growth by altering folate homeostasis. This appears unlikely, however, in view of the following observations: (1) the cytotoxic effects of CQS could not be reversed by folinic acid; (2) deoxyuridine suppression of thymidine incorporation was not affected by CQS treatment; (3) CQS did not inhibit dihydrofolate reductase from mammalian or bacterial sources; and (4) CQS toxicity in mice was not reduced by folinic acid. Experiments performed with analogues modified in the quinoxaline and para-amino phenyl functions indicated that tumor cell inhibition did not require preservation of the conventional sulfonamide structure.

Effectiveness of therapeutic anticoccidial drugs against recently isolated coccidia.

Litter samples were collected weekly from 17 broiler houses and coccidial oocysts were counted. Oocysts per gram were low on the Weeks 1 and 2 of the growout, increased to a peak on the Week 4, then declined to near zero on Weeks 6 and 7. Coccidia from each house were propagated and tested for sensitivity to therapeutic formulations of amprolium, sulfaquinoxaline, and sulfaquinoxaline plus pyrimethamine. The degree of control was based on the weight gains and lesion scores of the medicated groups in comparison with those of the unmedicated, infected, and uninfected groups. The predominant species found in the 17 isolates were Eimeria tenella and E. acervulina. Amprolium was effective against E. tenella in all isolates but only partly effective against E. acervulina. Sulfaquinoxaline and the potentiated mixture were effective against the E. acervulina but only partly effective against the E. tenella. The potentiated mixture was better against E. tenella than sulfaquinoxaline alone.

Anticoccidial drugs and duckling performance to four weeks of age.

Frequently, publications pertaining to waterfowl state that medicated feeds should not be fed to ducklings and goslings. In some localities, producers and hobbyists who raise a small number of ducklings and goslings can purchase only medicated chick, turkey, or gamebird starter and grower feeds. Because of the lack of documented information on this subject and the numerous requests for advice on this matter, anticoccidial drugs, zoalene, sulfaquinoxaline, and amprolium, were mixed in mash feed and fed to Khaki Campbell male ducklings to 4 weeks of age. No significant differences in mean body weight, mortality, and anatomical development were observed among the treatments. Medicated commercial crumble turkey and chick starter feeds produced significantly better feed conversion than the mash medicated or nonmedicated feeds. These differences can be attributed to greater feed spillage with the mash feed. Some ducklings in all treatments showed unsteadiness of gait and shaky legs. These conditions disappeared when the ducklings were moved from the battery brooder to an outside pen. Zoalene, sulfaquinoxaline, and amprolium used at the recommended levels for chickens and turkeys did not cause any leg or anatomical problems in ducklings.

Influence of some coccidiostatics on blood carotenoids and fatty acids in coccidiosis affected chicken.

Blood and liver carotenoids and A-vitamin and blood fatty acid modifications were observed in Eimeria tenella infested chicken. 125 chicken were alloted in 5 groups: 3 infested groups were given Amprol, Suldrazin and Clopindol respectively, in their diet. A control group was infested, a second one non-infested. Biochemical examinations were performed 10 days following treatment when chicken were slaughtered and samples collected. In control groups and in Amprol and Clopindol treated groups were seen significant decreases of liver and blood carotenoids; blood and liver A-vitamin levels were higher in Amprol treated chicken; in Suldrazin treated group no significant modifications could be shown as compared to the non-infested controls. Blood fatty acid levels were higher (43.5-47.8%) in the four infested groups and lower in the non-infested controls (39.4%). Non-saturated fatty acid levels varied within 51 and 56.4% in infested groups.

Mutagenicity screening of feed additives in the microbial system.

18 feed additives were tested for DNA-modifying effects by the repair test named "rec-assay" with Bacillus subtillis H17 (rec+) and M45 (rec-), and for mutagenicity with Escherichia coli WP2 hcr and 5 Salmonella typhimurium tester strains with the use of a top-agar overlay method. Carbadox, furazolidone, panazon and zoalene were positive in both assays. The former 3 were mutagenic for TA100, TA98 and WP2 hcr, while zoalene was mutagenic for all strains. These 4 compounds did not require a metabolic activation for their mutagenic activities. Nicarbazin was weakly mutagenic for TA1538 and TA98 with and without S9 mix. Amprolium and caprylohydroxamic acid also showed very weak mutagenicities only for TA100 with S9 mix and for WP2 hcr with and without S9 mix, resp. The mutagenic activities of carbadox, furazolidone and panazon for TA100 were reduced only by the addition of S9 mix, but not by S9 fraction or blood, whereas that of zoalene was decreased by any of the 3 factors.

Speciation studies with Eimeria acervulina and Eimeria mivati.

Eimeria mivati was described as a new species of chicken coccidia in 1964 by Edgar and Seibold, but recently some British workers have relegated its status to that of a variety of Eimeria acervulina. Using strains supplied by Dr. Edgar, we have prepared lines of E. acervulina resistant to methyl benzoquate, sulfaquinoxaline and robenidine and a line of E. mivati resistant to methyl benzoquate. Genetic transfer of resistance between the various lines of E. acervulina to produce doubly-resistant coccidia has been demonstrated, but no such transfer could be obtained between E. mivati resistant to methyl benzoquate and the resistant lines of E. acervulina. Although some immunological relationship between E. acervulina and E. mivati has been demonstrated, we conclude that this failure of the 2 organisms to interbreed lends support to the status of E. mivati as a distinct species.

Electrophoretic variation of enzymes: a further marker for genetic studies of the Eimeria.

Embryo-adapted strains of Eimeria mivati and E. mivati var. diminuta, differing in their sensitivity to sulphaquinoxaline and electrophoretic mobilities of lactate dehydrogenase, were crossed. E. mivati was sulphaquinoxaline-resistant and characterised by an electrophoretic form of the enzyme denoted lactate dehydrogenase-1 whereas E. mivati var. diminuta was sulphaquinoxaline-sensitive and characterised by lactate dehydrogenase-6. Progeny recovered from the cross were passaged in embryonating eggs given sulphaquinoxaline and the (drug-resistant) parasites recovered were characterised by both lactate dehydrogenase-1 and lactate dehydrogenase-6. Controls showed that those parasites characterised by the recombinant phenotype of drug-resistant and lactate dehydrogenase-6 had been produced by the cross-fertilisation of gametes.

The effect of sulfaquinoxaline feed medication on the immunologic response to a Pasteurella multocida vaccine administered to turkeys via drinking water.

Five groups of turkeys received C.U. strain Pasteurella multocida vaccine in the drinking water for one day. One group received the 0.1% level of sulfaquinoxaline feed medication at the time of vaccination, while 4 other groups received the feed medication 1, 2, 3 and 4 days after vaccination, respectively. Two weeks after vaccination all groups were exposed to a virulent P-1059 strain of P. multocida by the drinking water route. The results suggest that turkeys on the feed medication at the time of vaccination and possibly those receiving the initial fed medication the next day, failed to experience a satisfactory immunologic response. The possible ill effects of the sulfaquinoxaline feed medication on the duration of the immunity was not determined. A schedule and level of feed medication approved by the Food and Drug Administration was used; namely, 2 days on the 0.1% level, 3 days on a normal ration, and then 2 additional days on the 0.05% level of sulfaquinoxaline feed medication.

Transferred drug-resistance in Eimeria maxima.

A series of experiments is described in which two drug-resistant strains of Eimeria maxima were passaged together in untreated chicks. The resultant oocysts were then inoculated into chicks treated with both drugs. When strains resistant to methyl benzoquate and sulphaquinoxaline or clopidol and sulphaquinoxaline were used the resultant infections were not controlled by the double treatment, indicating the acquisition of resistance factors by one strain from the other. When strains resistant to clopidol and methyl benzoquate were used the phenomenon was not observed.

The effect of feeding diets containing permitted antibiotics on the faecal excretion of Salmonella typhimurium by experimentally infected chickens.

Groups of 45 chickens were fed continuously on diets containing 10 or 100 mg./kg. of different 'growth-promoting' antibiotics and infected orally with a nalidixic acid-resistant mutant of Salmonella typhimurium. The amount of S. typhimurium organisms excreted in their faeces was estimated by culturing them at weekly intervals and in a standard manner on plates of brilliant green agar containing sodium nalidixate, both direct and after enrichment in selenite broth. All of four groups fed diets containing 100 mg./kg. of nitrovin in three different experiments excreted much larger amounts of S. typhimurium than did groups fed antibiotic-free diets. In some, but not all, experiments, larger amounts were also excreted by groups fed diets containing 10 mg./kg. of nitrovin or 10 or 100 mg./kg. of flavomycin or tylosin. Feeding diets containing 10 or 100 mg./kg. of virginiamycin or bacitracin either did not influence or slightly increased the amount of S. typhimiurium excreted. Two groups fed continuously on diets containing 100 or 500 mg./kg. of sulphaquinoxaline for 44 days excreted smaller amounts of the S. typhimurium organisms that did groups fed antibiotic-free diets; no sulphonamide-resistant organisms of the S. typhimurium strain were isolated from the faeces.

Dietary and management procedures for development of consistent hemorrhagic symptoms in chicks.

Chicks hatched from S.C.W.L. pullets housed in laying cages and fed a diet without vitamin K were compared with chicks hatched from pullets fed a similar ration supplemented with vitamin K3 at 750 mcg./kg. of feed. Eggs were incubated after 4, 5 and 7 months vitamin K depletion. Chicks were fed a basal chick starter with no source of vitamin K or the basal supplemented with various levels of sulfaquinoxaline (SQ) (0.0125-0.050%) or with 750 mcg./kg. of vitamin K3. Blood clotting time was determined on chicks at the beginning and at weekly intervals during each experiment. Blood clotting time was significantly increased by a SQ feeding, but no hemorrhages developed in chicks hatched from hens fed the diet containing vitamin K. Day-old chicks hatched after 4 months vitamin K depletion showed elevated blood clotting times. There was no significant change in blood clotting time during the experiment for chicks fed the basal. Blood clotting time was significantly reduced in one week for chicks supplemented with vitamin K and increased for chicks fed 0.0375 or 0.050% SQ. These levels of SQ also resulted in 25 and 47.5% skin hemorrhages. Five months vitamin K depletion of breeder hens resulted in increased incidence of skin hemorrhages, but blood clotting times were similar to those at four months. Seven months vitamin K depletion of breeder hens resulted in increased blood clotting times as compared with 4 months and 100% incidence of hemorrhages in chicks fed rations containing 0.0375 or 0.050% SQ.