The comparative metabolism of diisopropyl methylphosphonate in mink and rats.

This study reports the metabolism of carbon-14labeled diisopropyl methylphosphonate (DIMP) in mink and rats, undertaken to better understand the dose-related mortality reported for mink in a previous study. In both male and female mink and rats, DIMP was rapidly absorbed after oral administration; it was metabolized by a saturable pathway to a single metabolite, isopropyl methylphosphonate (IMPA), which was rapidly excreted, primarily in the urine (90%). Fecal radioactivity, also identified as IMPA, was 1.7-3.1% of the administered dose. Female rats had a slower rate of conversion of DIMP to IMPA and less total excretion of IMPA than male rats. Metabolism of DIMP administered intravenously was not very different from that given orally in both species. These data indicate that mink absorb, metabolize, and excrete DIMP (as IMPA) in a manner very similar to mice, rats, and dogs.

Subchronic oral toxicity study of diisopropyl methylphosphonate in mink.

Diisopropyl methylphosphonate (DIMP), produced during manufacture of the chemical agent GB (Sarin), is a groundwater contaminant at Rocky Mountain Arsenal, Colorado. DIMP was fed for 90 days to dark brown "Ranch Wild" mink housed under controlled indoor conditions. One-year-old mink, 10 of each sex, were fed 0, 50, 450, 2700, 5400, or 8000 ppm in standard ranch diet. Actual DIMP consumption was 0, 8, 73, 400, 827, and 1136 mg/kg body wt/day, respectively. Two additional groups of 10 served as "pair-fed" controls. Body weight and food intake were recorded weekly. Complete blood count and 15 chemical analytes were measured at Weeks 0, 3, 7, and 13. Necropsy and microscopic examination were performed on all mink. No clinical morbidity or deaths occurred. Both sexes fed 8000 ppm ate approximately 20% less and weighed approximately 20% less than the controls; 5400 ppm females had a 10% weight decrement. Plasma cholinesterase (ChE) decreased in the top three dose groups starting at Week 3. At 13 weeks, decrements were approximately 50% but returned to normal after 1 week without DIMP. Erythrocyte ChE was not reduced. Heinz bodies occurred in 10-15% of RBCs in 50% of 8000 ppm mink at 13 weeks, and 0.1-2.0% of RBCs in 25% at 2700 ppm. There were mild decreases in RBC count, hematocrit, and hemoglobin, and increases in reticulocyte count, at the 5400 and 8000 ppm doses. All recovered within 3 weeks after DIMP was withdrawn. The 8000 ppm group had marginal splenic hematopoiesis, histologically. No other treatment-related changes were noted. The 450 ppm dose was a clear no-effect level (approximately 73 mg DIMP/kg body wt/day). Compared to reports of similar studies of DIMP in rats and dogs, these mink displayed no unique species susceptibility.

Functional aspects of Escherichia coli rep helicase in unwinding and replication of DNA.

The gene for Escherichia coli rep helicase (rep protein) was subcloned in a pBR plasmid and the protein overproduced in cells transformed with the hybrid DNA. The effect of purified enzyme on strand unwinding and DNA replication was investigated by electron microscopy. The templates used were partial duplexes of viral DNA from bacteriophage fd::Tn5 and reannealed DNA from bacteriophage Mu. The experiments with the two DNA species show DNA unwinding uncoupled from replication. The single-stranded phage fd::Tn5 DNA with the inverted repeat of transposon Tn5 could be completely replicated in the presence of the E. coli enzymes rep helicase, DNA binding protein I, RNA polymerase and DNA polymerase III holoenzyme. A block in the unwinding step increases secondary initiation events in single-stranded parts of the template, as DNA polymerase III holoenzyme cannot switch across the stem structure of the transposon.

Intermediate stages in enzymatic replication of bacteriophage fd duplex DNA.

Using purified enzymes, double strand replication of phage fd DNA has been dissected into several intermediate steps. (i) Phage fd gene 2 protein cleaves supercoiled phage fd replicative form at a specific site in the viral strand (Meyer, T. F., Geider, K., Kurz, C., and Schaller, H. (1979) Nature 278, 365-367). (ii) Relaxed covalently closed circular replicative form DNA which is also formed by gene 2 protein as a side product in the initiation reaction preceding replication is converted into supercoils by DNA gyrase. (iii) The enzyme forms a noncovalent complex at the generated nick that is necessary for initiation of subsequent unwinding. (iv) The Escherichia coli rep helicase (rep protein) and E. coli DNA binding protein I unwind the double-stranded DNA. (v) Concomitant DNA replication by E. coli DNA polymerase III holoenzyme results in the formation of rolling circle intermediates. The double-stranded core of the rolling circle remains in an open form, thus allowing continued synthesis during several rounds of replication. (vi) Processing of replicated viral DNA can be subdivided into the cleavage and the circularization of viral single strands. Comparative studies of fd and phi X174 replication in vitro have revealed differences in the kinetics of individual steps besides an apparent contrast in the conformation of rolling circle intermediates in the electron microscopy where fd DNA features extended tails rather than looped-back structures observed for phi X174 DNA.

Bacteriophage fd gene-2 protein. Processing of phage fd viral strands replicated by phage T7 enzymes.

Bacteriophage T7 gene 4 protein and DNA polymerase of the phage were used to study the viral strand synthesis of bacteriophage fd in vitro. Cleavage of supercoiled phage fd replicative form (RF) by fd gene 2 protein produced a nick at a specific site in the viral strand. The cleaved double-stranded DNA was unwound by T7 gene 4 protein and T7 DNA polymerase and the 3' end of the nicked strand simultaneously extended according to the rolling circle mechanism. After a complete round of DNA synthesis fd gene 2 protein cleaved the viral strand presumably at the same site, where the endonuclease cuts fd RF I, and subsequently sealed the single-stranded linear DNA into a circle. The reaction products were analyzed by velocity sedimentation, gel electrophoresis and electron microscopy. Most of the single-stranded DNA synthesized was circular. No host proteins were required for the formation of the single-stranded circles. Strand switching of the T7 DNA polymerase indicated by double-stranded tails of the rolling circle structures reduced the yield of viral single-stranded circles in this enzyme system.

Replication of phase fd RF with fd gene 2 protein and phage T4 enzymes.

Bacteriophage fd replicative form DNA with a nick in the viral strand serves as a template for DNa replication with purified bacteriophage T4 enzymes. As anticipated from previous in vitro studies carried out with this system (Morris, C. F., Sinha, N. K., and Alberts, B. M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4800-4804), DNA is synthesized by a rolling circle mechanism. We show here that the DNA strands synthesized are processed by the phage fd gene 2 protein into unit length products, providing that the gene 2 protein is present at the moment when this DNA is made. The products are mostly unit length linear single strands, indicating that the circularization step normally catalyzed by gene 2 protein subsequent to its site-specific cleavage of an fd DNA strand occurs only inefficiently in this system. The gene 2 protein reduces the level of DNA synthesis by 2-fold at low concentrations, even though it only cleaves the DNA products efficiently at higher levels of the enzyme. This indicates that there are at least two different effects of the fd gene 2 protein in processing of viral fd DNA.

Metabolism, distribution and anticonvulsant properties of N,N'- dimethoxymethyl-phenobarbital in the rat.

The in vivo metabolism of N,N'-dimethoxymethyl phenobarbital (DMMP) and the anticonvulsant properties of its metabolites were studied in the rat. At 10 and 30 minutes after i.p. administration, DMMP (ethyl-1-14C) represented less than 3% of plasma radioactivity, whereas N-monomethoxymethyl phenobarbital (MMP) was 78 and 75%, respectively, phenobarbital (PB) 12 and 20%, apparent mephobarbital 6 and 2% and less than 5% of the radioactivity remained at the origin. Peak levels of MMP were reached at 30 minutes in liver and 60 minutes in plasma and brain. At 4 hours, MMP had declined to 7% in plasma and was not detected in brain while PB rose to 93% of total 14C in plasma and brain. SKF-525A blocked (90%) the in vivo conversion of DMMP to MMP and completely inhibited the formation of PB, apparent mephobarbital and unidentified polar metabolites. MMP after oral administration was effective against maximum electroshock seizures at a time when brain levels of PB derived from MMP were insufficient to account for the total observed protection. However, MMP appears less potent than PB against pentylenetetrazol seizures.

The effects of 5,5-diphenylbarbituric acid on experimental seizures in rats: correlation between plasma and brain concentrations and anticonvulsant activity.

Diphenylbarbituric acid was administered by gavage to male albino Sprague-Dawley rats for evaluation of anticonvulsant activity and analysis of blood plasma and brain drug concentrations. A method for gas-liquid chromatographic analysis of blood plasma and brain for diphenylbarbituric acid is described. The drug was effective in the maximum electroshock test and as an antagonist to clonic seizures and death produced by pentylenetetrazol. Brain concentrations associated with 50 to 70% protection in the maximum electroshock seizure test were of the order of 2 to 6 mug/g of brain and are thus equal to values for phenobarbital in this test. Large doses of diphenylbarbituric acid (up to 2,800 mg/kg) failed to produce signs of neurotoxicity. The results of the present investigation continue to indicate that diphenylbarbituric acid exhibits promise as a potential antiepileptic agent.

Clinical evaluation of eterobarb, a new anticonvulsant drug.

Two clinical investigations of a new anticonvulsant, eterobarb, N,N' dimethoxymethyl phenobarbital (DMMP), were conducted in separate geographic regions. The drug has considerable efficacy in reducing the frequency of partial seizures, with and without secondary generalization, as well as generalized tonic-clonic seizures. Sedation did not appear to be as prominent with this barbiturate as it was with phenobarbital or primidone.