Biodegradation of selected aminophosphonates by the bacterial isolate Ochrobactrum sp. BTU1.

Aminophosphonates, like glyphosate (GS) or metal chelators such as ethylenediaminetetra(methylenephosphonic acid) (EDTMP), are released on a large scale worldwide. Here, we have characterized a bacterial strain capable of degrading synthetic aminophosphonates. The strain was isolated from LC/MS standard solution. Genome sequencing indicated that the strain belongs to the genus Ochrobactrum. Whole-genome classification using pyANI software to compute a pairwise ANI and other metrics between Brucella assemblies and Ochrobactrum contigs revealed that the bacterial strain is designated as Ochrobactrum sp. BTU1. Degradation batch tests with Ochrobactrum sp. BTU1 and the selected aminophosphonates GS, EDTMP, aminomethylphosphonic acid (AMPA), iminodi(methylene-phosphonic) (IDMP) and ethylaminobis(methylenephosphonic) acid (EABMP) showed that the strain can use all phosphonates as sole phosphorus source during phosphorus starvation. The highest growth rate was achieved with AMPA, while EDTMP and GS were least supportive for growth. Proteome analysis revealed that GS degradation is promoted by C-P lyase via the sarcosine pathway, i.e., initial cleavage at the C-P bond. We also identified C-P lyase to be responsible for degradation of EDTMP, EABMP, IDMP and AMPA. However, the identification of the metabolite ethylenediaminetri(methylenephosphonic acid) via LC/MS analysis in the test medium during EDTMP degradation indicates a different initial cleavage step as compared to GS. For EDTMP, it is evident that the initial cleavage occurs at the C-N bond. The detection of different key enzymes at regulated levels, form the bacterial proteoms during EDTMP exposure, further supports this finding. This study illustrates that widely used and structurally more complex aminophosphonates can be degraded by Ochrobactrum sp. BTU1 via the well-known degradation pathways but with different initial cleavage strategy compared to GS.

Synthesis and evaluation of p-iodo-phentermine (IP) as a brain perfusion imaging agent.

p-(123I and 131I)iodo alpha,alpha-dimethylphenethylamine (p-iodophentermine, IP) as the alpha-methylated analogue of iodoamphetamine has been prepared. It is hoped that this methyl substitution will increase the lipophilicity of the agent, enhance resistance to metabolism by monoamine oxidase, and will result in increased initial uptake and slower washout from the brain as compared to N-isopropyl-p-(123I)iodoamphetamine. IP was prepared by diazotization of p-aminophentermine followed by decomposition of the diazonium salt with KI. Radioiodinated IP was prepared either by the solid-phase isotopic exchange reaction or by decomposition of the piperidinotriazene derivative with a radiochemical yield of 40-60%. Biodistribution of 131I-IP in rats showed brain uptake in the range of 1.7% dose g-1 at 5, 30 and 60 min. Imaging studies with 123I-IP in dogs showed high brain extraction and slow washout of activity.

Chlorphentermine-induced alterations in the lungs of vitamin E-deficient and -supplemented rats: quantitative ultrastructural analysis of Type II Cell response.

Response of alveolar Type II (T-II) cells of male Sprague-Dawley weanling rats maintained on vitamin E (VE)-deficient and -supplemented diets to chlorphentermine (CP) treatment was evaluated. Ultrastructural and quantitative procedures were used to assess the T-II cell-drug interaction. Subjective examination of photomicrographs revealed numerous disrupted surfactant lamellar bodies in the CP-treated animals, as compared to fewer in controls. In addition, an unusual amorphous material was associated with these cells in drug-treated animals. Quantitative assessment confirmed the above subjective impression and revealed an increase in the size of T-II cells in treated rats. Morphometric evaluation showed that CP treatment significantly altered the volume density of cytoplasm, mitochondria, and rough endoplasmic reticulum (RER). Furthermore, a significant interaction between the diet and treatment with respect to volume fractions of mitochondria, RER, and Golgi complex was observed. The present study indicates that cytoplasmic components in T-II cells are altered as a result of CP treatment and that VE status of the animals influences the nature and extent of these alterations.

Chlorphentermine-induced alterations in the lungs of vitamin E-deficient and supplemented rats: 1. Biochemical and morphometric analysis of the pulmonary response.

Male Sprague-Dawley rats were maintained on diets containing 0.60, or 300 ppm vitamin E (VE) for 9 weeks with chlorphentermine (CP) or saline vehicle (SV) treatments administered over the last 3 weeks (20 mg CP/kg for one week and 30 mg CP/kg for subsequent 2 weeks or equivalent volume of saline vehicle). Spontaneous erythrocyte hemolysis averaged 68% for VE-deficient (0 ppm) and less than 9% for VE-supplemented (60 and 300 ppm) animals prior to saline and CP treatments. These values were not changed significantly by vehicle or drug administration. The lung-to-body weight ratios nearly doubled and the total lung phospholipid levels increased equivalently (three- to fourfold) in all three CP-treated VE groups as compared to corresponding SV controls. The levels of thiobarbituric acid-reactive material (TBA-RM), an index of lipid peroxidation, was increased in the lung tissue above SV controls in all dietary groups with the deficient group being the highest. There was no difference in the TBA-RM values of 60 and 300 ppm groups within each group. Quantitative morphometric analysis revealed that in the VE-supplemented groups, CP treatment caused a significant increase in the number of alveolar macrophage foam cells (FC) with a slight increase in the volume density of surfactant-like material (SLM). By comparison, there were fewer FCs but a larger quantity of SLM in the VE-deficient group. The results suggest that VE deficiency modifies the pulmonary response to CP resulting in lipid peroxidation-induced FC disintegration and the accumulation of SLM.

Impairment in pulmonary bioenergetics following chlorphentermine administration to rats.

Biochemical alteration in pulmonary oxidative metabolism and morphological integrity of lung mitochondria were examined in rats following administration of chlorphentermine (30 mg/kg, ip, 5 days per week) for 1 or 2 weeks. During the first week of treatment, body weight gain and food intake were decreased markedly but returned to control levels during the second week. Phospholipid content of the lung was increased 31% and 110% after 1 and 2 weeks of treatment, respectively. This was accompanied by a striking intraalveolar accumulation of hypertrophic alveolar macrophages. The metabolism of both (1-14C)- and (6-14C)-glucose was decreased 27% and 26%, respectively, after 2 weeks of drug treatment. In rat lung mitochondria, chlorphentermine significantly lowered the RCR and ADP/O ratio and stimulated state 4 respiration. State 3 respiration and uncoupled state respiration were unaffected. These data indicate that chlorphentermine functions as a true uncoupler of oxidative phosphorylation when administered in vivo. Furthermore, disruption of mitochondrial membranes was observed frequently in lung mitochondria from treated animals. These combined data indicate that the induction of pulmonary phospholipidosis by chlorphentermine is accompanied by marked alterations in subcellular bioenergetics and mitochondrial structure.

Generalized lipidosis in newborn rats and Guinea pigs induced during prenatal development by administration of amphiphilic drugs to pregnant animals.

Pregnant rats and guinea pigs were treated throughout the second half of gestation with amphiphilic drugs (chlorphentermine, chlorcyclizine, chloroquine) known to induce generalized lipidosis. The offspring were sacrificed immediately after birth, and several tissues (lung, liver, kidney, spleen, pituitary gland, adrenal gland, spinal cord, hypothalamus) were examined by electron microscopy. Generalized lipidosis was found in the offspring of both species, albeit of lesser degree than in the mothers. The results show that fetal and adult tissues respond to lipidosis-inducing drugs in a qualitatively similar way; the quantitative differences found may be related to pharmacokinetic and cellular factors.

Morphological and biochemical alterations of the lung after application of chlorphentermine.

The influence of short-term and chronic administration of chlorphentermine (Chlph) on the structure and phospholipid (PL)-content of the lungs was studied in guinea pigs. The drug was given daily in a dose of 40 mg per kg body weight i.p. The PL-content of the whole lung as well as the phosphatidyl choline (PC)-portion were estimated in animals treated with Chlph for 4, 5 and 6 weeks. In a further experiment, the influence of 2, 4 and 28 days application on the PL-content of the lung lavage fluid separated into the acellular and cellular (macrophage) fraction was examined. Morphologic studies of lung, liver, heart, kidney, spleen, brain, and pancreas supplemented the PL-content analysis. The amount of PL in the whole lung increases continuously with the duration of the treatment but only moderately. The PC-fraction of the total PL remains constant throughout the experiment. A substantially greater increase was detected in the lung lavage fluid whereby the cellular fraction is most affected. Very important is the result that the PL-content of the lung lavage fluid reaches its highest level already after two Chlph-injections. After short-term administration the morphological examination of the lung mainly shows inflammatory infiltrations, whereas after long-term application a foam cell reaction is mainly localized in the interstitium. The studies show that an increase of the amount of lung sufactant in guinea pigs is possible following Chlph administration.