Changes in DNA fragmentation during sperm preparation for intracytoplasmic sperm injection over time.

OBJECTIVE: To compare the DNA fragmentation of semen samples established by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) after incubation in polyvinylpyrrolidone (PVP) and hyaluronic acid (HA) for different time periods. DESIGN: Comparative prospective study. SETTING: Center for reproductive medicine. PATIENT(S): Twenty-seven semen samples from infertile patients. INTERVENTION(S): None. METHODS: Semen analysis and DNA fragmentation assays (TUNEL) were performed. Two groups were established: A) normal TUNEL (<20%); and B) Abnormal TUNEL (≥ 20%). TUNEL was performed in neat (T0), postgradient (TG), 1-hour postgradient (TG1), and 2-hour postgradient (TG2) samples and in TG2 samples after 0.5, 1.0, and 1.5 hours of incubation in PVP or HA. RESULT(S): TUNEL levels were significantly reduced after gradient separation compared with neat values. In group A, TUNEL levels were significantly higher in the TG2 + 1.5 hours in PVP and HA samples but did not reach abnormal levels. In group B, TUNEL levels were significantly higher in the TG2 + 1 hour in PVP and HA samples. CONCLUSION(S): Sperm DNA fragmentation significantly decreased after centrifugation gradient, regardless of the initial levels of the sample. Samples with TUNEL ≥ 20% were more susceptible to a significant increase in DNA fragmentation over time, with similar increases being observed over time for samples that were incubated in HA or PVP. These data may be relevant for sperm preparation for intracytoplasmic sperm injection.

Inclusion of the insecticide fenitrothion in dimethylated-ß-cyclodextrin: unusual guest disorder in the solid state and efficient retardation of the hydrolysis rate of the complexed guest in alkaline solution.

An anhydrous 1:1 crystalline inclusion complex between the organophosphorus insecticide fenitrothion [O,O-dimethyl O-(3-methyl-4-nitrophenyl)phosphorothioate] and the host compound heptakis(2,6-di-O-methyl)-ß-cyclodextrin (DIMEB) was prepared and its structure elucidated by single-crystal X-ray diffraction. This revealed two independent host molecules in the asymmetric unit. In one of these, the cavity is occupied by two disordered guest components (distinguishable as rotamers with respect to the P-OAr bond) while in the other, three distinct guest components with site-occupancies 0.44, 0.29 and 0.27 appear, the last having a reversed orientation relative to all the other components. Kinetic studies of the alkaline hydrolysis of fenitrothion in the presence of DIMEB showed a remarkable reduction of 84% in the rate of this reaction relative to that for the free substrate, a value exceeding those previously attained with the native hosts, ß- and γ-cyclodextrin, and fully methylated ß-cyclodextrin.

Effect of cyclodextrins on the reactivity of fenitrothion.

The hydrolysis reaction of fenitrothion was studied in water containing 2% dioxane and in the presence of native cyclodextrins (α-, ß- and γ-CD) and two commercially available modified derivatives, namely, permethylated ß- and α-cyclodextrin (TRIMEB and TRIMEA, respectively). The kinetics of the reaction in the presence of TRIMEA could not be measured because the complex formed is insoluble and precipitated even at low concentration. On the other hand, the reaction is only weakly affected by the presence of α-CD. The hydrolysis reaction is inhibited by all the other cyclodextrins. From the kinetic data the association equilibrium constants for the formation of the 1:1 inclusion complexes were determined as 417, 511 and 99M(-1) for ß-CD, TRIMEB and γ-CD, respectively. Despite the differences in the association constants for ß- and γ-CD, the observed inhibition effect is about the same and this is due to the fact that the rate of hydrolysis in the cavity of γ-CD is smaller than that in the cavity of ß-CD. The strongest inhibitor is TRIMEB and this result is consistent with the known structure of the complex in the solid state.

Reactivity of the insecticide fenitrothion toward O and N nucleophiles.

The reactivity of Fenitrothion (1) toward several O- and N-based nucleophiles, including ambident and alpha-nucleophiles, was investigated in basic media at 25 degrees C in water containing 2% 1,4-dioxane. In the reactions with HO(-) and HOO(-) quantitative formation of 3-methyl-4-nitrophenoxide (2) was observed indicating a S(N)2(P) pathway. In the reactions with NH(2)OH, NH(2)O(-), and BuNH(2), demethylfenitrothion (4) was formed along with 2, indicating competition between the S(N)2(P) and S(N)2(C) pathways; no evidence of a S(N)Ar pathway was observed in any case. The observed rate constants were dissected into the values corresponding to the S(N)2(P) and S(N)2(C) pathways. The yield of 4 depends on the nucleophile and on the pH of the reaction, being the main product in the case of BuNH(2). With HOO(-), NH(2)OH, and NH(2)O(-) a significant alpha-effect was observed, confirming the participation of the nucleophile in the rate-limiting step of the reaction.

Solid-state structures and thermal properties of inclusion complexes of the organophosphate insecticide fenitrothion with permethylated cyclodextrins.

The X-ray crystal structures and thermal stabilities of the inclusion complexes formed between the organophosphate insecticide fenitrothion [O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate] and the host compounds TRIMEA and TRIMEB (permethylated alpha- and beta-cyclodextrins, respectively) are reported. In the complex (TRIMEA)(2).fenitrothion 1, the guest phosphate ester group is disordered and the molecule is fully encapsulated within a novel TRIMEA dimer in which the secondary rims of the two host molecules are in close contact. In contrast, the complex TRIMEB.fenitrothion 2 is monomeric and the guest molecule is statistically disordered over two positions, with the phosphate group inserted in the host cavity in both cases. Thermal analysis indicated gradual and partial loss of the guest in 1 during heating between 130 degrees C and the melting point of the complex (approximately 200 degrees C), whereas complex 2 displayed significant mass loss only after fusion of the complex at 161 degrees C.

Investigation of the inclusion of the herbicide metobromuron in native cyclodextrins by powder X-ray diffraction and isothermal titration calorimetry.

We report the formation of inclusion complexes between the phenylurea herbicide metobromuron [3-(p-bromophenyl)-1-methoxy-1-methylurea] and beta- and gamma-cyclodextrin in the solid state. Formation of crystalline inclusion complexes by the kneading method was confirmed by powder X-ray diffraction and further structural characterization using the principles of isostructurality followed. In addition, DeltaH degrees , DeltaS degrees , DeltaG degrees and the association constants (K) at 298K were determined for complex formation in solution using isothermal titration calorimetry. The magnitudes of K for the formation of 1:1 complexes between metobromuron and alpha-, beta- and gamma-CD were estimated as 598, 310 and 114, respectively.