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)     

2,4-D and alkaloid accumulation in periwinkle cell suspensions

Omission of 2,4-D from culture medium during one subculture of Catharanthus roseus cells, strain C20, resulted in an increased alkaloid accumulation, without effect on growth. Alkaloid accumulation, rather than growth, seemed to be more sensitive to 2,4-D. 2,4-D inhibited alkaloid accumulation essentially during growth phase, but its inhibitory effect during this period was partially reversible. As this reversibility was underlined only during the stationary phase, this suggested that this action could be situated upstream in a terpenoid non-specific pathway. 2,4-D feeding showed that inhibition is weaker and weaker as the alkaloid accumulation period proceeds. Auxin action during this period could take place downstream in specific alkaloid pathways. The lower alkaloid accumulation obtained after loganic acid feeding compared to that obtained with secologanin and loganin could indicate that loganic acid methylation should be one of the 2,4-D target(s).     

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 calcium and magnesium with the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae

The activity of the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae was measured in the presence of increasing concentrations of magnesium and calcium. Calcium pyrophosphate (dissociation constant Kd = 1.9 microM) inhibited pyrophosphatase by competition with magnesium pyrophosphate (Kd = 50 microM). The small movements of calcium detected in mitochondria from yeast may be physiologically significant for the control of inorganic pyrophosphatase activity and the concentration of pyrophosphate in the matrix of yeast mitochondria.     

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.     

Interactions of mitochondrial ATP synthesis and the creatine kinase equilibrium in skeletal muscle

This study aimed to evaluate the effect of FR128998, (1s,6s)-1-benzyl-10-(3-pyridyl-methyl)-7-thia-10-azaspiro [5,6]-dodecan-11-one 7,7-dioxide hydrochloride, a novel platelet activating factor (PAF) receptor antagonist, on endotoxin lipopolysaccharide-induced disseminated intravascular coagulation in rats. Experimental disseminated intravascular coagulation was induced by an infusion of lipopolysaccharide at 0.25 mg/kg/h for 4 h. Simultaneous infusion of FR128998 (0.25 and 1.0 mg/kg/h) with lipopolysaccharide dose dependently inhibited thrombocytopenia, but not leukopenia. The changes in coagulation parameters of disseminated intravascular coagulation, i.e., prolongation of activated partial thromboplastin time and elevated levels of fibrinogen/fibrin degradation products, were also prevented by the treatment with FR128998. In addition, FR128998 attenuated the increase in serum tumor necrosis factor (TNF) which appeared during the initial stage of disseminated intravascular coagulation. FR128998 (10 microM) also inhibited the TNF production by peripheral blood leukocytes or alveolar macrophages stimulated by lipopolysaccharide in vitro. Furthermore, TNF production induced by PAF itself in vitro was also inhibited in the presence of FR128998. These data indicate that PAF plays a pivotal role in the development of disseminated intravascular coagulation via TNF production.     

Interactions of cyclophilin with the mitochondrial inner membrane and regulation of the permeability transition pore, and cyclosporin A-sensitive channel

Mammalian mitochondria possess an inner membrane channel, the permeability transition pore (MTP), which can be inhibited by nanomolar concentrations of cyclosporin (CS) A. The molecular basis for MTP inhibition by CSA remains unclear. Mitochondria also possess a matrix cyclophilin (CyP) with a unique N-terminal sequence (CyP-M). To test the hypothesis that it interacts with the MTP, we have studied the interactions of CyP-M with rat liver mitochondria by Western blotting with a specific antibody against its unique N terminus. Although sonication in isotonic sucrose at pH 7.4 refraction sediments with submitochondrial particles at 150,000 x g. We show that the interactions of this CyP-M pool with submitochondrial particles are disrupted (i) by the addition of CSA, which inhibits the pore, but not of CSH, which does not, and (ii) by acidic pH condition, which also leads to selective inhibition of the MTP; furthermore, we show that the effect of acidic pH on CyP-M fully prevents the inhibitory effect of H+ on the MTP (Nicolli, A., Petronilli, V., and Bernardi, P. (1993) Biochemistry 32, 4461-4465). These data suggest that CyP-M inhibition by CSA and protons may be due to unbinding of CyP-M from its putative binding site on the MTP. A role for CyP-M in MTP regulation is also supported by a study with a series of CSA derivatives with graded affinity for CyP. We show that with each derivative the isomerase activity of CyP-M purified to homogeneity is similar to that displayed at inhibition of MTP opening, CyP-M (but not CyP-A) and decreased efficiency at MTP inhibition is obtained by substitution in position 8 while a 4-substituted, nonimmunosuppressive derivative is a as effective as the native CSA molecule, indicating that calcineurin is not involved in MTP inhibition by CSA.     

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.     

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.     

Structural determinants of substrates and inhibitors: probing glutamate transporters with 2,4-methanopyrrolidine-2,4-dicarboxylate

Using an intramolecular [2 + 2] photocyclization, 2,4-methanopyrrolidine-2,4-dicarboxylate was prepared as a conformationally locked analogue of glutamate. This compound, in combination with two other pyrrolidine dicarboxylates, has been used to define the structural elements that differentiate substrate and nonsubstrate inhibitors of a high-affinity, sodium-dependent glutamate transporter.     

Interactions between metallothionein inducers in rat liver and primary cultures of rat hepatocytes

The interaction of Zn, stress and endotoxin on liver metallothionein (MT) regulation has been studied in the rat. Zn, stress and endotoxin increased liver MT levels significantly, by 12-, 5- and 8-fold, respectively. The previous administration of Zn to stress or endotoxin treatments increased MT levels by 35- and 42-fold, respectively, indicating a synergistic effect in both cases. In contrast, when liver MT was preinduced by stress, MT levels were further increased by endotoxin only in an additive manner. In another experiment where liver MT induction by stress was studied in control rats and in rats with preinduced MT by Zn, endotoxin or stress, it was found that Zn pretreated animals had higher MT-I mRNA levels than endotoxin- or stress-pretreated ones. No synergisms between dexamethasone, Zn, TNF and IFN were observed in primary culture of hepatocytes. These results suggest that the observed synergisms between Zn and other MT inducers in vivo in the liver is a consequence of increased Zn levels in the body and mobilization capacity, with concomitant MT synthesis.     

Interactions of Titanium and Phosphorus with Tungsten

XRD has been applied to the component interaction in the Ti – W – P system, and an isothermal section at 1070 K has been constructed for the phase diagram in the range 0-0.67 mole fraction of P. A substitutional solid solution based on the phosphide Ti₃P is formed: Ti_3.0-2.4W_0-0.6P, a = 0.9953(2)-0.9934(3) nm, c = 0.4994(2)-0.4943(2) nm. It is confirmed that the ternary phosphide Ti_0.5W_0.5P is present of NbAs structure type. Interactions in the titanium – VIa metal – phosphorus systems are analyzed.     

Interactions of indomethacin with lysosomes and proteins

The stabilizing action of indomethacin on lysosomes and protein in vivtro was studied. The magnitude of the stabilizing action on lysosomes was greater than that of aspirin or oxyphenbutazone, similar to that of phenylbutazone, but less than that of prednisolone. The stabilizing action of indomethacin on lysosomes possibly may contribute to its anti-inflammatory properties.     

A mechanism for ethanol-induced damage to liver mitochondrial structure and function

Mitochondria isolated from rats chronically fed ethanol demonstrated a marked inability to produce energy. The respiratory control ratio, the ADP/O ratio and state 3 respiration rates were all decreased. Coupled with other data, a progression of ethanol-induced changes is proposed with site I being altered prior to site II. Quantitation of mitochondrial cytochromes revealed decreases in cytochromes b and aa3 and an increase in c1. Evaluation of respiration activity in relation to temperature showed ethanol-induced changes in the transition temperature (Tf) which may have been related to changes in the lipid composition of the inner membrane. Mitochondrial membranes were separated, and analysis of fatty acids and phospholipids was performed. Various fatty acids were altered in both membranes; however, the outer membrane was altered more severely. A decrease in the arachidonate : linoleate ratio was observed only in the outer membrane; however, there was no ethanol-induced change in degree of unsaturation in either membrane. Phospholipid quantitation showed a reduction of total lipid phosphorous/mg protein in both membrane fractions; however, the inner membrane was most affected. Cardiolipin was the only phospholipid in this membrane which remained unaltered. The evidence indicates that the mechanism for ethanol-induced damage to the liver mitochondrion involves lipid compositional changes as well as changes in cytochromes and possibly other proteins.