Accumulation and metabolism of drugs and CYP probe substrates in zebrafish larvae.

This study examined the accumulation and metabolism of a number of drugs and commonly used probes for human cytochrome P450s (CYPs) in zebrafish larvae under conditions relevant to pharmacological and toxicological assays. Studies with cisapride, chlorpromazine, verapamil, testosterone, and dextromethorphan showed that the zebrafish larvae catalyze a range of phase 1 (oxidation, N-demethylation, O-de-ethylation, and N-dealkylation) and phase 2 (sulfation and glucuronidation) reactions. Both similarities and differences in the metabolic pathways were observed in zebrafish larvae when compared to mammals. Metabolism of phenacetin to paracetamol and dextromethorphan to dextrorphan (metabolic reactions catalyzed by CYP 1A2 and 2D6 in humans respectively) were observed in the zebrafish larvae. In addition the zebrafish larvae 7 days post fertilization (7 d.p.f.) hydroxylated diclofenac, bupropion, tacrine, and testosterone. Although metabolites of several compounds were detected in zebrafish larvae, in the instances where the metabolite amounts were quantified, the amount of any specific metabolite formed was low, accounting for only a small percentage of the amount of parent compound added. Furthermore, when the concentrations of metabolite present in the zebrafish larvae were compared with the measured level of parent compound, the metabolite concentrations were always much lower than that of parent compound. Overall, for the compounds used in the current study it is unlikely that the quantified metabolites would significantly contribute to the outcome of safety pharmacology or toxicology studies conducted in zebrafish larvae under the paradigms typically used for such investigations.

Zebrafish: an emerging technology for in vivo pharmacological assessment to identify potential safety liabilities in early drug discovery.

The zebrafish is a well-established model organism used in developmental biology. In the last decade, this technology has been extended to the generation of high-value knowledge on safety risks of novel drugs. Indeed, the larval zebrafish appear to combine advantages of whole organism phenotypic assays and those (rapid production of results with minimal resource engagement) of in vitro high-throughput screening techniques. Thus, if appropriately evaluated, it can offer undeniable advantages in drug discovery for identification of target and off-target effects. Here, we review some applications of zebrafish to identify potential safety liabilities, particularly before lead/candidate selection. For instance, zebrafish cardiovascular system can be used to reveal decreases in heart rate and atrial-ventricular dissociation, which may signal human ether-a-go-go-related gene (hERG) channel blockade. Another main area of interest is the CNS, where zebrafish behavioural assays have been and are further being developed into screening platforms for assessment of locomotor activity, convulsant and proconvulsant liability, cognitive impairment, drug dependence potential and impaired visual and auditory functions. Zebrafish also offer interesting possibilities for evaluating effects on bone density and gastrointestinal function. Furthermore, available knowledge of the renal system in larval zebrafish can allow identification of potential safety issues of drug candidates on this often neglected area in early development platforms. Although additional validation is certainly needed, the zebrafish is emerging as a versatile in vivo animal model to identify off-target effects that need investigation and further clarification early in the drug discovery process to reduce the current, high degree of attrition in development.

Cervical cytology biobanking: quality of DNA from archival cervical Pap-stained smears.

AIMS: Cervical cytology biobanking is a feasible concept in cervical pathology and could be an indispensable tool for fundamental and applied molecular biological research. PCR is a powerful molecular technique that can be performed on a variety of cervical sample types including Pap-stained cervical smears. However, since the quality of DNA from such specimens is inferior to that from fresh tissue, the correct processing methods are required. This study evaluates three commercial isolation methods and one digestion procedure for their ability to obtain DNA suitable for PCR from fixed and stained Pap smears. METHODS: The High Pure PCR Template Preparation kit, the NucliSENS easyMAG system, the QIAamp DNA Mini Kit and crude proteinase K digestion were used to obtain DNA for subsequent PCR applications. Amplification of beta-globin was performed to verify the presence and integrity of target DNA. The influence of PCR inhibitors and extent of DNA fragmentation were analysed. RESULTS: All commercial DNA isolation techniques provided DNA suitable for PCR amplification, and DNA isolated from 10-year-old archival smears yielded amplicons up to 400 base pairs. Conversely, crude proteinase K digestion limited the amplicon size to 300 bp and did not consistently yield amplifiable digests, as these were contaminated with PCR-inhibiting factors and debris. CONCLUSION: The study indicates that commercial DNA isolation techniques are suitable for PCR amplification of DNA isolated from archival smears, yielding amplicons up to 400 base pairs. Proteinase K digestion is not suitable to obtain amplifiable DNA from fixed and stained Pap-stained smears.

Differential expression of the GTL2 gene within the callipyge region of ovine chromosome 18.

The inheritance pattern of the skeletal muscle hypertrophy phenotype caused by the callipyge gene has been characterized as polar overdominance. We hypothesized that this trait may be caused by a gain or loss of gene expression because of the reversible nature of the phenotype in paternal vs. maternal inheritance. Suppression subtraction cDNA probes were made from skeletal muscle mRNA of normal (NN) and callipyge (C(Pat)N(Mat)) animals and hybridized to Southern blots containing bacterial artificial chromosomes (BACs) that comprise a physical contig of the callipyge region. The CN-NN probes hybridized to two ovine and seven bovine BACs. Sequence analysis of fragments within those BACs indicated short regions of similarity to mouse gene trap locus (gtl2). Northern blots analysis of RNA from hypertrophy-responsive muscles show a population of GTL2 mRNA centred around 2.4 kb that were abundantly expressed in 14-day prenatal NN and C(Pat)N(Mat) lambs but were down-regulated in day 14 and day 56 postnatal NN lambs. The expression of GTL2 remained elevated in 14- and 56-day-old C(Pat)N(Mat) lambs as well as in 56-day-old N(Pat)C(Mat) and CC lambs. Expression of GTL2 in the supraspinatus, which does not undergo hypertrophy, was very low for all genotypes and ages. Isolation of cDNA sequences show extensive alternative splicing and a lack of codon bias suggesting that GTL2 does not encode a protein. The mutation of the callipyge allele has altered postnatal expression of GTL2 in muscles that undergo hypertrophy and will help identify mechanisms involved in growth, genomic imprinting and polar overdominance.

Human-ovine comparative sequencing of a 250-kb imprinted domain encompassing the callipyge (clpg) locus and identification of six imprinted transcripts: DLK1, DAT, GTL2, PEG11, antiPEG11, and MEG8.

Two ovine BAC clones and a connecting long-range PCR product, jointly spanning approximately 250 kb and representing most of the MULGE5-OY3 marker interval known to contain the clpg locus, were completely sequenced. The resulting genomic sequence was aligned with its human ortholog and extensively annotated. Six transcripts, four of which were novel, were predicted to originate from within the analyzed region and their existence confirmed experimentally: DLK1, DAT, GTL2, PEG11, antiPEG11, and MEG8. RT-PCR experiments performed on a range of tissues sampled from an 8-wk-old animal demonstrated the preferential expression of all six transcripts in skeletal muscle, which suggests that they are under control of common regulatory elements. The six transcripts were also shown to be subject to parental imprinting: DLK1, DAT, and PEG11 were shown to be paternally expressed and GTL2, antiPEG11, and MEG8 to be maternally expressed.

Fine-mapping and construction of a bovine contig spanning the ovine callipyge locus.

The callipyge (CLPG) gene was fine-mapped by linkage analysis to a 4.6-cM chromosome interval on distal ovine OAR18q, flanked by microsatellite markers IDVGA30 and OY3. The OAR18q linkage map and human HSA14q transcript map were aligned by genotyping two bovine-hamster whole-genome radiation hybrid panels with the microsatellite markers, as well as with sequences corresponding to HSA 14q genes. Using Type I loci mapping to the IDVGA30-OY3 interval as anchor points, we have constructed a 1.4-Mb bovine BAC contig containing the IDVGA30-OY3 interval. We demonstrate that the IDVGA30-OY3 interval spans approximately 770 kb and contains at least four genes: YY1, WARS, DLK1, and GTL2.

Construction and characterization of an ovine BAC contig spanning the callipyge locus.

We describe the construction of an ovine BAC contig spanning a 4.6 centimorgan (cM) chromosome segment known to contain the callipyge (CLPG) locus. The contig comprises 21 ovine BAC clones jointly covering approximately 900 kilobases (Kb). Two gaps in the BAC contig, spanning 10 and 7.5 Kb, respectively, were bridged by long range PCR. The corresponding chromosome region was shown to be characterized by an unusually low Kb to cM ratio (164 Kb/cM) and a high density of Not1 sites (1:126 Kb) possibly reflecting a high gene density in the corresponding chromosome region. Equivalent amplification of 64 sequence tagged sites spanning the corresponding region from homozygous +/+ and CLPG/CLPG individuals disproves the hypothesis of a major deletion causing the CLPG mutation.

The callipyge phenomenon: evidence for unusual genetic inheritance.

In 1983, a male lamb exhibiting a pronounced muscular hypertrophy, particularly noticeable in the hind quarters, was born into a commercial Dorset flock in Oklahoma. The ram was premonitorily called Solid Gold. He subsequently produced offspring expressing the unusual phenotype, which is referred to as callipyge (Greek: calli- beautiful + -pyge buttocks). Animals demonstrating the callipyge phenotype are all descendants of this founder ram. These animals produce leaner, higher yielding carcasses, but there is some concern with decreased tenderness of the loin. Genetic characterization of the locus has demonstrated a unique mode of inheritance termed polar overdominance, in which only heterozygous offspring inheriting the mutation from their sire express the phenotype. The three other genotypes are normal in appearance. Progeny data indicate that reactivation of the maternal callipyge allele occurs after passage through the male germ line, although this reactivation is not absolute. The callipyge gene has been mapped to the distal end of ovine chromosome 18.

Polar overdominance at the ovine callipyge locus.

An inheritable muscular hypertrophy was recently described in sheep and shown to be determined by the callipyge gene mapped to ovine chromosome 18. Here, the callipyge phenotype was found to be characterized by a nonmendelian inheritance pattern, referred to as polar overdominance, where only heterozygous individuals having inherited the callipyge mutation from their sire express the phenotype. The possible role of parental imprinting in the determinism of polar overdominance is envisaged.