Interaction of 2,4-dichlorophenoxyacetic acid (2,4-D) with cell and model membranes

2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, is a component of the "agent orange' whose toxicity has been extensively studied without definite conclusions. In order to evaluate its perturbing effect upon cell membranes, 2,4-D was made to interact with human erythrocytes and molecular models. These studies were performed by scanning electron microscopy on red cells, fluorescence spectroscopy on dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles and X-ray diffraction on multilayers of DMPC and dimyristoylphosphatidylethanolamine (DMPE). It was observed that 2,4-D induced a pronounced shape change to the erythrocytes. This effect is explained by the herbicide interaction with the outer monolayer of the red cell membrane.     

Genetic and phenotypic diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria isolated from 2,4-D-treated field soils

Forty-seven numerically dominant 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated at different times from 1989 through 1992 from eight agricultural plots (3.6 by 9.1 m) which were either not treated with 2,4-D or treated with 2,4-D at three different concentrations. Isolates were obtained from the most dilute positive most-probable-number tubes inoculated with soil samples from the different plots on seven sampling dates over the 3-year period. The isolates were compared by using fatty acid methyl ester (FAME) profiles, chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences, and hybridization patterns obtained with probes for the tfd genes of plasmid pJP4 and a probe (Spa probe) that detects a distinctly different 2,4-D-degrading isolate, Sphingomonas paucimobilis (formerly Pseudomonas paucimobilis). A total of 57% of the isolates were identified to the species level by the FAME analysis, and these isolates were strains of Sphingomonas, Pseudomonas, or Alcaligenes species. Hybridization analysis revealed four groups. Group I strains, which exhibited sequence homology with tfdA, -B, -C, and -D genes, were rather diverse, as determined by both the FAME analysis and the REP-PCR analysis. Group II, which exhibited homology only with the tfdA gene, was a small group and was probably a subset of group I. All group I and II strains had plasmids. Hybridization analysis revealed that the tfd genes were located on plasmids in 75% of these strains and on the chromosome or a large plasmid in the other 25% of the strains. One strain exhibited tfdA and -B hybridization associated with a plasmid band, while tfdC and -D hybridized with the chromosomal band area. The group III strains exhibited no detectable homology to tfd genes but hybridized to the Spa probe. The members of this group were tightly clustered as determined by both the FAME analysis and the REP-PCR analysis, were distinctly different from group I strains as determined by the FAME analysis, and had very few plasmids; this group contained more of the 47 isolates than any other group. The group III strains were identified as S. paucimobilis. The group IV strains, which hybridized to neither the tft prove nor the Spa probe, were as diverse as the group I strains as determined by the FAME and REP-PCR analyses. Most of group IV strains could not be identified by the FAME analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of herbicides 2,4-D and dinoseb with liver mitochondrial bioenergetics

The use of drugs for common pregnancy complications like premature labor, hypertensive diseases, and premature rupture of membranes with chorioamnionitis is reviewed. In addition, new publications on antiviral drugs in HIV-positive pregnant patients are also discussed. Among the drugs, suppressing premature labor side-effects of beta-mimetics are of growing concern. The effectiveness of other agents like magnesium, indomethacin, and sulindac are addressed. The various mechanisms explaining the beneficial effect of magnesium in pre-eclampsia are reviewed and new data on antihypertensives, such as labetalol, calcium channel blockers, and methyldopa are presented. The evidence from various clinical trials on the value of low-dose aspirin as a prophylactic agent against pregnancy-induced hypertension, pre-eclampsia, and intrauterine growth retardation in high-risk and low-risk patients is compared. Pharmacokinetic data including transplacental transfer of antibiotics and anti-HIV nucleosides are part of this review.     

Metabolic alterations in hepatocytes promoted by the herbicides paraquat, dinoseb and 2,4-D

The cytotoxic effects of the herbicides paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride), dinoseb (2-sec-butyl-4,6-dinitrophenol) and 2,4-D (2,4-dichlorophenoxyacetic acid) on freshly isolated rat hepatocytes were investigated. Paraquat and 2,4-D (1-10 mM) caused a dose and time dependent cell death accompanied by depletion of intracellular glutathione (GSH) and mirroring increase of oxidized glutathione (GSSG). Dinoseb, the most effective cytotoxic compound under study (used in concentrations 1000 fold lower than paraquat and 2,4-D), exhibited moderate effects upon the level of GSH and GSSG. These limited effects are at variance with significant effects upon the adenine and pyridine nucleotide contents. ATP and NADH levels are rapidly depleted by herbicide metabolism. This depletion is observed in the millimolar range for paraquat and 2,4-D and in the micromolar range for dinoseb. 2,4-D completely depletes cellular ATP, with subsequent cell death, as detected by LDH leakage. Paraquat rapidly depletes NADH, according to the redox cycling of the herbicide metabolism. The most effective compound is dinoseb since it exerts similar effects as described for paraquat and 2,4-D at concentrations 1000 fold lower. Simultaneously with NADH and ATP depletion, the levels of ADP, AMP and NAD+ increase in hepatocytes incubated in the presence of the herbicides. In contrast to NADH, the time course and extent of ATP depletion and fall in energy charge correlate reasonably with the time of onset and rate of cell death. It is concluded that the herbicides, paraquat and 2,4-D are hepatotoxic and initiate the process of cell death by decreasing cellular GSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H NMR determination of intracellular volume in cell suspensions

A 1H NMR method has been developed for determining the intracellular and extracellular volumes in a cell suspension. The method is quick, simple, and inexpensive. A comparison of the ratios of the water and Tris buffer resonances in a cell suspension and in a buffer solution gives the intracellular volume. The most important precaution to take is to ensure that coil loading is identical in both solutions and that the NMR signal is not saturating. The method was validated with a 20% polyethylene glycol solution. A comparison with radiolabel methods for volume determination found that the radiolabel probe of extracellular volume did not penetrate the cell wall water of Enterococcus faecalis, resulting in an overestimation of the intracellular volume, and that tritiated water probably exchanged with macromolecules, causing an underestimation of intracellular volume.     

Pollution of the agricultural lands of Bashkortostan by the herbicide 2,4-D and dioxins]

Market forces present the nursing profession with an urgency to prepare gerontological nurses to assume significant roles in the managed care industry. An understanding of the current managed care environment underscores the need for training. Nurses require a "managed care" skill-set encompassing a firm grasp of the organization, financing, delivery, and policy implications of managed care as well as advanced practice clinical skills and a sound business orientation. The importance of the consumer as a significant player in managed care is highlighted.     

Factors inducing mast cell accumulation in skeletal muscle

It has been suggested that mast cells contribute to the phenotype of dystrophinopathies, but the mechanisms of their recruitment into the skeletal muscle remain hypothetical. The aim of this study is to quantify the presence of mast cells in muscle during the cellular events of myofibre degeneration and regeneration. For this purpose, we compare the mast cell profile in dystrophin-deficient mdx mice in which muscles exhibit spontaneous cycles of degeneration-regeneration from 3 weeks of age, with that in Swiss mice in which muscles were injured either by ischaemia or by notexin injection. Notexin is an A2-type phospholipase that rapidly disrupts myofibre plasma membranes, while ischaemia results in a slower process of degeneration. Both lesions are followed by a successful regeneration. In intact muscles, mast cell counts (mean +/- SEM/mm2) range from 1.8 +/- 1 to 4.3 +/- 1.6. The injection of notexin is far more potent in recruiting mast cells into damaged muscle than is ischaemia (118.5 +/- 13.0 vs 12.3 +/- 1.8/mm2). Thus we conclude that the early disruption of the myofibre membrane could elicit mast cell accumulation in skeletal muscle. This may explain the elevated number of mast cells observed in mdx muscles, as dystrophin deficiency is though to induce myofibre membrane leakage. On the other hand, mast cells are more numerous in muscles of young and adult mdx mice that are allowed to regenerate, than in muscles of older animals in which there is little regeneration and fibrosis develops. In injured muscles, the peak of mast cell number is at the onset of regeneration (by day 3 after notexin injection, and by day 11 after ischaemia), rather than during the phase of myofibre necrosis. Therefore, we suggest that the mast cells, through the effects of released mediators, could contribute to muscle regeneration.     

Enzyme immunoassay based survey of precipitation and surface water for the presence of atrazine, metolachlor and 2,4-D

The suitability of enzyme immunoassay (EIA) as a method of analysis for 2,4-D, atrazine and metolachlor contamination in water samples was determined by comparing EIA results to gas chromatography (GC) results. The comparison of EIA and GC results yielded a correlation coefficient of 0.92, 0.98 and 0.92 for 2,4-D, atrazine and metolachlor, respectively. EIA was used to monitor seasonal trends in the concentrations of 2,4-D, atrazine and metolachlor in surface water and precipitation throughout the province of Ontario, Canada. 2,4-D was detected in excess of 4 micrograms/L in urban creeks during the period of application. Concentrations of 43 and 9 micrograms/L of atrazine and metolachlor, respectively, were detected during the field application period in surface water samples from the Kintore Creek watershed. The levels of 2,4-D, atrazine and metolachlor detected exceeded the Canadian Water Quality Guidelines for the protection of fresh water aquatic life. Concentrations as high as 445 and 322 ng/L of atrazine and metolachlor, respectively, were detected in precipitation samples collected from 17 locations in Ontario during the herbicide application period. The EIA was shown to be qualitatively and quantitatively comparable to GC analysis.     

Dependence of microwave produced heating of cell suspensions on their concentration

This study compared three radionuclide techniques in distinguishing musculoskeletal infection from noninfectious inflammation. METHODS: Thirty-five orthopedic patients with suspected musculoskeletal infection were examined using three radionuclide techniques in sequence: triphasic bone scintigraphy, 99mTc radioleukocytes (99mTc-WBC) scintigraphy and 99mTc human immunoglobulin (99mTc-Hig) scintigraphy. Two "early" and "late" acquisitions were performed, at 4-6 hr and 20-24 hr postinjection, respectively. Patients who were diagnosed as suffering from noninflammatory lesions became the controls. We calcu"late"d for all studies one index of inflammation (Infl) as the ratio between counts in the uptake area and counts in an equal area of normal tissue. RESULTS: The "early" radiolabeled leukocytes and "late" Hig scintigraphy allowed the greatest ability to distinguish between infections and noninfectious inflammations (p < 0.011 and p < 0.016) with a sensitivity of 96.6% and 96.5% and specificity of 71% and 100%, respectively. Hig and radioleukocytes allowed distinguishing infections from noninflammatory diseases at both examinations. CONCLUSION: The "early" radioleukocyte scintigraphy allowed us to separate infections from noninfectious inflammations. In contrast, the same result can be obtained only with the "late" scan in the Hig study, but Hig mapped the spread of the inflammation into soft tissues better. Hig might be an alternative to radioleukocytes because of its simple preparation, similar accuracy and safety.     

Measurement of water transport during freezing in cell suspensions using a differential scanning calorimeter

A new technique using a differential scanning calorimeter (DSC) was developed to obtain dynamic and quantitative water transport data in cell suspensions during freezing. The model system investigated was a nonattached spherical lymphocyte (Epstein-Barr virus transformed, EBVT) human cell line. Data from the technique show that the initial heat release of a prenucleated sample containing osmotically active cells in media is greater than the final heat release of an identical sample of osmotically inactive or lysed cells in media. The total integrated magnitude of this difference, Deltaqdsc, was found to be proportional to the cytocrit and hence also to the supercooled water volume in the sample. Further, the normalized fractional integrated heat release difference as a function of temperature, Deltaq(T)dsc/Deltaqdsc, was shown to correlate with the amount of supercooled cellular water which had exosmosed from the cell as a function of subzero temperature at constant cooling rates of 5, 10, and 20 degrees C/min. Several important limitations of the technique are (1) that it requires a priori knowledge of geometric parameters such as the surface area, initial volume, and osmotically inactive cell volume and (2) that the technique alone cannot determine whether the heat released from supercooled cellular water is due to dehydration or intracellular ice formation. Cryomicroscopy was used to address these limitations. The initial cell volume and surface area were obtained directly whereas a Boyle-van't Hoff (BVH) plot was constructed to obtain the osmotically inactive cell volume Vb. Curve fitting the BVH data assuming linear osmometric behavior yielded Vb = 0.258V0; however, nonlinearity in the data suggests that the EBVT lymphocyte cells are not "ideal osmometers" at low subzero temperatures and created some uncertainty in the actual value of Vb. Cryomicroscopy further confirmed that dehydration was the predominant biophysical response of the cells over the range of cooling rates investigated. One notable exception occurred at a rate of 20 degrees C/min where evidence for intracellular ice formation due to a DSC measured heat release between -30 and -34 degrees C correlated with a higher end volume but no darkening of the cells during cryomicroscopy. For the cooling rate tested (5 degrees C/min) the cryomicroscopy data correlated statistically very well with the DSC water transport data. A model of water transport was fit to the DSC water transport data and the average (5, 10, and 20 degrees C/min) biophysical parameters for the EBVT lymphocytes were found to be Lpg = 0.10 micro m/min-atm, ELp = 15.5 kcal/mol. Finally, the decrease in heat release from osmotically active cells measured by the DSC during repetitive freezing and thawing was found to correlate strongly with the viability of the cells measured during identical freeze/thaw protocols with cryomicroscopy. This shows the additional ability of the technique to assess freeze/thaw injury. In summary, this DSC technique is a promising new approach for measuring water transport in cellular systems during freezing.     

Molecular genetic analysis of the response of three soil microbial communities to the application of 2,4-D

The responses of three different soil microbial communities to the experimental application of 2,4-dichlorophenoxyacetic acid (2,4-D) were evaluated with a variety of molecular genetic techniques. Two of the three soil communities had histories of prior direct exposure to 2,4-D, and one had no prior direct application of any herbicide. Dominant 2,4-D degrading strains isolated from these soils the previous year were screened for hybridization with three catabolic genes (tfdA, tfdAII, and tfdB) cloned from the well-studied 2,4-D degradative plasmid, pJP4, revealing varying degrees of similarity with the three genes. Hybridization of total community DNA from the three soils with the tfd gene probes also indicated that pJP4-like tfd genes were not harboured by a significant percentage of the community. Community level response was evaluated by the comparison of different treatments by Random Amplified Polymorphic DNA (RAPD) fingerprints and by community DNA cross-hybridization. No differences between treatments within the same soil were detected in any of the RAPD fingerprints generated with 17 primers. Community DNA cross-hybridization also indicated that the application of 2,4-D at the applied rates did not quantitatively affect the structure of the soil microbial communities present in the three soils during the time-frame studied.     

In situ measurements of Doppler power vs. flow turbulence intensity in red cell suspensions

Whereas previous studies have shown that ultrasonic backscatter and Doppler power from blood are affected by flow turbulence, turbulence level has only been inferred from the flow Reynolds number and not directly measured. In this study, both ultrasonic Doppler power and flow turbulence intensity were measured in situ to quantify the relationship between Doppler power and flow turbulence. Three grid meshes of different geometries were used in a steady-flow mock loop to generate controlled levels of flow turbulence in porcine red blood cell saline suspensions. Doppler power was measured by a 10-MHz PW Doppler flowmeter, and the turbulence intensity by using constant-temperature hot film anemometry. We showed that Doppler power is affected by turbulence and hematocrit in a complex way. At a fixed hematocrit, Doppler power increases nonlinearly with turbulence intensity and, at fixed turbulence intensity, Doppler power peaks at an optimal hematocrit level that increases with turbulence level. The shape factor, introduced by Lucas and Twersky (1987) to take into account effects of shape and orientation of the scatterers in a dense distribution of small and tenuous scatterers, was estimated by fitting the experimental data to the theoretical model. The results indicate that shape factor decreases with increasing turbulence intensity.     

Protoporphyrin IX accumulation in cells treated with 5-aminolevulinic acid: dependence on cell density, cell size and cell cycle

NO and cGMP have emerged as important signal transduction mediators of the effects of certain hormones, inter-/intracellular signals, toxins and drugs. However, a major challenge is to define relevant criteria for determining which of the many NO and/or cGMP effects are dependent on cGMP-dependent protein kinases (cGKs). Important criteria include that: (1) the cell types/tissues investigated contain at least one form of cGK which is activated by the cGMP-elevating agent in the intact cell system; (2) specific activators/inhibitors of cGKs mimic/block the effects of cGMP-elevating agents in the intact cell system; and (3) the cGMP effect is absent or blunted in cGK-deficient systems, or can be reconstituted by the introduction of active cGKs. Previously, analysis of cGK activity in intact cells has been very difficult. However, the analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation by polyclonal antibodies and newly developed monoclonal antibodies, each of which specifically recognize different phosphorylation sites, allows the quantitative measurement of cGK activity in intact cells. With the use of these methods, the properties of certain cGK mutants, cGK activators (cGMP, 8-Br-cGMP, 8-pCPT-cGMP) as well as various "specific cGK inhibitors" (KT 5823, Rp-8Br-PET-cGMPS, Rp-8-pCPT-cGMPS, H8 and H89) were investigated. Although these "specific cGK inhibitors" have been widely used to establish or rule out functional roles of cGKs, very few studies have actually addressed the efficiency/specificity of such compounds in intact cells. Our results demonstrate that these inhibitors are useful tools only when used in combination with other experimental approaches and biochemical evidence.