Subchronic 3-month oral toxicity study of grape seed and grape skin extracts.

Meganatural brand grape seed extract (GSE) and grape skin extract (GSKE), containing proanthocyanidin (PAC) polyphenolic compounds, are intended for use in food as functional ingredients exhibiting antioxidant activity. Proanthocyanidins, as well as the minor constituent phenolic compounds in GSE and GSKE, are present naturally in many foods such as fruits, vegetables, chocolate, tea, etc., and on average people consume 460-1000 mg/day of these combined substances. Although humans have ingested PACs for centuries without reported adverse effects, the current toxicology literature contains relatively little formal evidence regarding their safety. Accordingly, as part of a program to investigate the safety of GSE and GSKE, these products were incorporated into chow and fed to rats for at least 3 months in a GLP-compliant subchronic toxicity study. Groups of CD (Sprague-Dawley) Crl:CD IGS BR rats (20 males and 20 females per group) were fed diets containing GSE at concentrations of 0, 0.63, 1.25 or 2.5% (w/w); GSKE was fed at 2.5% (w/w) only. Clinical observations were recorded and body weight and feed consumption measured throughout the study. After 1 month, blood was obtained from 10 rats/sex/group by retrobulbar puncture for interim measurement of clinical pathology. At the end of the study the rats were subjected to a full necropsy, aortic blood samples were collected for clinical pathology, selected organs were weighed and a complete list of tissues was preserved from all animals. Histologic examination was performed on all tissues from control and high-dose GSE and GSKE groups. There were no treatment-related changes that were considered to be of toxicologic significance. Therefore, a dietary concentration of 2.5% GSE or 2.5% GSKE was considered to be a no-observed-adverse effect level (NOAEL). This was equivalent to a time-weighted average dose over the course of the study of approximately 1.78 g/kg body weight/day GSE or GSKE in male rats and 2.15 g/kg body weight/day in female rats.

Cutaneous vasoproliferative lesions in goats.

Abstract. In a retrospective survey of caprine neoplastic disease, eight masses were diagnosed as cutaneous vascular tumors. The typical clinical presentation was a solitary raised, bleeding mass. No predilection with regard to age, breed, sex, or anatomic location was found. Reevaluation of the microscopic features of the masses resulted in diagnoses of hamartoma (2), hemangioma (4), and hemangiosarcoma (2). An endothelial cell origin was confirmed in all seven tumors tested immunohistochemically for factor VIII-related antigen. Although rarely reported, goats display a range of cutaneous vascular growth abnormalities similar to those observed in other domestic animals.

Caprine genital leiomyosarcoma.

An aged Saanen doe was euthanized following repeated severe hemorrhage from the vulva. Necropsy examination revealed mural thickening of tubular genitalia with firm, protruding intralumenal masses containing blood-filled cavitations, and effacement of normal cervical architecture. Histologically, uterine and cervical masses comprised a variably dense population of mildly pleomorphic spindle cells forming interlacing streams supported by variably dense collagenous stroma. Immunoperoxidase staining of neoplastic cells was positive for muscle-specific actin, supporting the diagnosis of low-grade leiomyosarcoma. Months later, the doe's twin was likewise euthanized due to persistent bleeding from the vulva associated with a large vulvar mass having histopathologic features similar to those of the previous case. The clinical, gross, and histologic findings are similar to five cases of caprine genital leiomyosarcoma identified in retrospectively analyzed case material. Analysis of caprine tumor accessions over 20 years demonstrated a significantly higher incidence of genital leiomyosarcoma within the Saanen breed.

A deletion mutation causes hemophilia B in Lhasa Apso dogs.

Hemophilia B is a bleeding disorder caused by a deficiency of clotting factor IX (FIX). A colony of FIX deficient Lhasa Apso dogs has been established and the molecular basis of hemophilia B has been determined. The plasma factor IX levels were < 1% of normal canine levels in affected dogs. A complex deletion mutation at nucleotides 772-777 was found when hepatocyte cDNA from a hemophilia B dog was sequenced. The sequence was identical to the normal canine sequence except for a deletion including nucleotides 772-776 and a C-->T transition at nucleotide 777. The mutation results in mRNA instability and a premature termination codon in the nucleotide sequence encoding the activation peptide. The mutation was verified by sequencing genomic DNA from an FIX-deficient dog. A genetic test for the detection of heterozygous animals was established using heteroduplex analysis. Although hemophilia B has been described in many dog breeds, this is only the second mutation to be sequenced. The Lhasa Apso dog model should be valuable for evaluating novel strategies for treating hemophilia B such as gene therapy.

Genetic test for pyruvate kinase deficiency of Basenjis.

Pyruvate kinase (PK) deficiency is an autosomal, recessive, inherited disease of Basenjis that causes chronic, regenerative, hemolytic anemia. Diagnostic methods currently used to identify carrier animals rely on measurement of erythrocyte PK activity and frequently give equivocal results. A genetic test incorporating polymerase chain reaction amplification of genomic DNA and restriction fragment length polymorphism has been developed to determine the PK genotype of Basenjis. To determine whether results of this genetic test compared with results of standard tests for PK deficiency, erythrocyte PK activity, hematocrit, and reticulocyte counts were determined in, and the genetic test was performed on, 24 dogs. The genetic test accurately identified the 11 dogs whose PK genotype was known prior to this study, and results were consistent with results of measuring erythrocyte PK activity in the remaining 13 dogs. The genetic test may be of value in determining PK genotype of Basenjis.

The molecular basis of canine pyruvate kinase deficiency.

Inherited hemolytic anemia due to pyruvate kinase (PK) deficiency is an autosomal recessive disease of the Basenji dog that closely resembles human PK deficiency. Characterization of transcriptional and translational expression of PK isozymes and sequencing of DNA from normal and mutant dogs were performed to identify the genetic defect in Basenji dogs. Measurement of erythrocytic PK activity by ion exchange chromatography, substrate kinetics, immunologic reactivity, and electrophoretic mobility suggests that M2-type PK is the major form of PK activity in erythrocytes of PK-deficient dogs, in contrast to normal dogs having only R-type PK activity. Both R-type and M2-type PK mRNA are detectable in reticulocytes of PK-deficient dogs, suggesting that the aberrant isozyme expression is not due to a failure in the erythroid maturational switch from M2- to R-type isozymes. Nucleotide sequence data from wild-type and mutant R-type PK cDNA identified a single nucleotide deletion, delta C433, in the mutant cDNA. The deduced amino acid sequence predicts a truncated mutant protein devoid of all residues contributing to the catalytic site of the wild-type protein. In the absence of R-type PK activity, there is anomalous compensatory expression of M2-type PK in erythroid cells of PK-deficient Basenjis. The PK-deficient Basenji dog may be valuable in somatic cell gene therapy trials involving manipulation of hematopoietic stem cells.

Effect of long-term tetracycline exposure (drinking water additive) on antibiotic-resistance of aerobic gram-negative intestinal flora of rats.

A study was undertaken to determine the effect of 2 years of intermittent administration of tetracycline in drinking water on antibiotic resistance in the aerobic gram-negative enterobacteria of rats in a closed colony. The bacterial isolates examined were resistant to tetracycline and streptomycin. Minimal inhibitory concentrations of tetracycline and streptomycin for intestinal organisms were similar in all of the animals, regardless of whether the animals were sampled while they were given drinking water with added tetracycline or at intervals of 3, 8, and 9 months after the antibiotic was no longer added to the drinking water. Biochemical examination of the isolates from each principal showed that Escherichia coli was the predominant enteric organism. In conjugation experiments, all E coli and Klebsiella pneumoniae isolated transferred tetracycline and streptomycin resistance to an E coli K-12 recipient. Four different strains of rats that had not been treated with tetracycline (controls) were examined for tetracycline resistance. Tetracycline-resistant Proteus mirabilis was isolated from the intestines of these animals. Plasmid-mediated resistance could not be demonstrated. The E coli and P vulgaris isolates from these control animals were susceptible to tetracycline.