Metabolism studies of the antischistosomal drug praziquantel using tandem mass spectrometry: qualitative identification of 17 hydroxylated metabolites from mouse urine.

Schistosomiasis is a parasitic liver infection which is known to affect many aspects of drug metabolism. Praziquantel (PZQ) is the drug of choice for treating this disease. PZQ is known to be highly metabolized, but the effect of the disease on its metabolism has not been investigated. Control mice and mice infected with Schistosoma mansoni were dosed with PZQ and their urines were examined for the presence of metabolites using a triple-quadrupole mass spectrometer (tandem mass spectrometer). The collisionally induced dissociation of PZQ was remarkable in its structurally significant fragments. From this we were able to identify 17 hydroxylated metabolites of PZQ from purified urine samples without further chemical separation, including three monohydroxylated, six dihydroxylated, and eight trihydroxylated metabolites. There were no qualitative differences in metabolite production between control and infected animals.

Metabolism studies of the antischistosomal drug praziquantel using tandem mass spectrometry: distribution of parent drug and ten metabolites obtained from control and schistosome-infected mouse urine.

The collisionally activated dissociation spectrum of the antischistosomal drug praziquantel (PZQ) has many structure-specific fragmentations which permit identification of PZQ and seventeen of its hydroxylated metabolites in mouse urine. These fragmentations may also be used to quantify the metabolic pattern of PZQ. In the present study, a triple-quadrupole mass spectrometer system has been used to generate [M + H]+ ions for PZQ and its monohydroxy, dihydroxy and trihydroxy metabolites which yield daughter ions capable of quantifying PZQ and ten of these metabolites. The goal of these experiments was to evaluate the effect of this hepatic infection on drug metabolism. This was accomplished in two steps. First, the amount of unmetabolized, mono- and dihydroxylated PZQ was established from the [M + H]+ ions. Then the specific metabolites at each level of hydroxylation were determined from daughter ion spectra. The product of these two values produces the metabolite pattern. The reproducibility of these assays ranged from good, with a coefficient of variation of 3% for the most abundant metabolite, to poor (43%) for PZQ, which is only 1% of the total elimination pattern. The excretion of unchanged PZQ and two dihydroxylated metabolites was enhanced in animals bearing schistosomiasis compared to control mice, while a third dihydroxylated metabolite was depressed.

Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins.

Organization and evolution of the alcohol dehydrogenase gene in Drosophila.

The alcohol dehydrogenase (Adh) gene was isolated from Drosophila simulans and D. mauritiana, and the DNA sequence of a 4.6-kb region, containing the structural gene and flanking sequence, was determined for each. These sequences were compared with the Adh region of D. melanogaster to characterize changes that occur in the Drosophila genome during evolution and to identify conserved sequences of functional importance. Drosophila simulans and D. mauritiana Adh are organized in a manner similar to that of D. melanogaster Adh, including the presence of two promoters for the single Adh gene. This study identified conserved flanking elements that, in conjunction with other studies, suggest regions that may be involved in the control of Adh expression. Inter- and intraspecies comparisons revealed differences in the kinds of sequence changes that have accumulated. Sequence divergence in and around the Adh gene was used to assess inter- and intraspecies evolutionary relationships. Finally, there appears to be an unrelated structural gene located directly 3' of the Adh transcribed region.

Assignment of the human and mouse prion protein genes to homologous chromosomes.

Purified preparations of scrapie prions contain one major macromolecule, designated prion protein (PrP). Genes encoding PrP are found in normal animals and humans but not within the infectious particles. The PrP gene was assigned to human chromosome 20 and the corresponding mouse chromosome 2 using somatic cell hybrids. In situ hybridization studies mapped the human PrP gene to band 20p12----pter. Our results should lead to studies of genetic loci syntenic with the PrP gene, which may play a role in the pathogenesis of prion diseases or other degenerative neurologic disorders.

Hepatic drug metabolism in rats with experimental chronic renal failure.

The activities of several hepatic microsomal, mitochondrial, and cytosolic drug-metabolizing enzymes, as well as the components of the cytochrome P-450 system, were determined in vitro for control, sham-operated, and uremic rats. Chronic renal failure (CRF) was produced by a two-stage surgical procedure. In this model, the animals were maintained for 21 days postoperatively before assay. During this time, serum urea nitrogen (SUN) levels rose from control levels of 21 mg/dl to an average of 63 mg/dl. Enzymes assayed included microsomal N-, O-, and S-demethylases, esterase, and UDP-glucuronyl transferase; monoamine oxidase; and alcohol dehydrogenase. CRF caused decreases of 24-32% in N- and O-demethylase activities, while S-demethylase, esterase, UDP-glucuronyl transferase, and monoamine oxidase activities were not altered significantly. Alcohol dehydrogenase activity was increased 71%. In addition, the functional components of the microsomal mixed-function oxidase system were assayed. CRF caused a 26% decrease in cytochrome P-450 levels, as compared to sham-operated controls, but cytochrome b5 and NADPH-cytochrome c (P-450) reductase were not altered. CRF caused an increase in hexobarbital sleeping time of more than 7-fold. In each case, alterations in enzyme activities or cytochrome P-450 correlated with the extent of renal failure, as determined by elevated SUN levels.

Nucleotide sequence comparison of the Adh gene in three drosophilids.

The alcohol dehydrogenase (Adh) gene has been isolated from Drosophila simulans and D. mauritiana by screening lambda clone libraries of each with a previously cloned Adh gene from D. melanogaster. The isolated lambda clones were subcloned and partially sequenced to determine the relatedness of these species and to examine details of evolutionary change in the structure of the Adh gene. We report the sequence of the first 704 nucleotides of each gene as well as 127 bases in the 5' untranslated region. When these sequences are compared, D. melanogaster differs from D. simulans and D. mauritiana by 2.8% and 3.1%, respectively. D. simulans and D. mauritiana differ by only 1.8%, implying that they are more closely related to each other than either is to D. melanogaster. This is consistent with phylogenetic relationships established by a variety of genetic, biochemical, and morphological means and illustrates that DNA sequencing of a single gene may be used to assess the evolutionary relationships of species.

Evolution of single-copy DNA and the ADH gene in seven drosophilids.

Single-copy DNA was isolated from Drosophila melanogaster and hybridized with total genomic DNA of D. melanogaster, D. mauritiana, D. simulans, D. pseudoobscura, D. willistoni, D. hydei and D. virilis. The duplexes were thermally eluted from hydroxyapatite and the data used to assess the relatedness of each species to D. melanogaster. The general pattern of relatedness was similar to that predicted by morphological methods but with some notable exceptions. The rate of nucleotide substitution was estimated to be greater than 0.66% of bases per million years. An unexpected, rapidly evolving component of D. melanogaster single-copy DNA was identified. The relatedness of these species was also studied with respect to the gene coding for alcohol dehydrogenase (ADH). The ADH gene, previously cloned from D. melanogaster (Goldberg 1980), was hybridized with Southern blots of genomic digests of the seven species. The intensity and position of the hybridizing bands suggest the amount of divergence of the gene. Divergence was quantitated by reassociation of a fragment of the cloned ADH gene with total DNA of the seven drosophilids and thermal elution of the resultant duplexes from hydroxyapatite. The ADH gene was isolated from genomic clone libraries of D. melanogaster, D. simulans and D. mauritiana and further studied by comparison of position of restriction sites. Species relationships deduced from comparison of total single-copy DNA and the ADH gene were consistent, demonstrating that a single gene can reflect divergence of the entire genome.

Characterization of five members of the actin gene family in the sea urchin.

Hybridization of an actin cDNA clone (pSA38) to restriction enzyme digests of Strongylocentrotus purpuratus DNA indicates that the sea urchin genome contains at least five different actin genes. A sea urchin genomic clone library was screened for recombinants which hydridize to pSA38 and four genomic clones were isolated. Restriction maps were generated which indicate that three of these recombinants contain different actin genes, and that the fourth may be an allele to one of these. The restriction maps suggest that one clone contains two linked actin genes. This fact, which was confirmed by heteroduplex analysis, indicates that the actin gene family may be clustered. The linked genes are oriented in the same direction and spaced about 8.0 kilobases apart. In heteroduplexes between genomic clones two intervening sequences were seen. Significant homology is confined to the actin coding region and does not include any flanking sequence. Southern blot analysis reveals that repetitive DNA sequences are found in the region of the actin genes.