Veterinary Medicine Commons^™

The Future Of Teratology Research Is In Vitro, Jarrod Bailey, Andrew Knight, Jonathan Balcombe

Jarrod Bailey, PhD

Birth defects induced by maternal exposure to exogenous agents during pregnancy are preventable, if the agents themselves can be identified and avoided. Billions of dollars and manhours have been dedicated to animal-based discovery and characterisation methods over decades. We show here, via a comprehensive systematic review and analysis of this data, that these methods constitute questionable science and pose a hazard to humans. Mean positive and negative predictivities barely exceed 50%; discordance among the species used is substantial; reliable extrapolation from animal data to humans is impossible, and virtually all known human teratogens have so far been identified in spite …

Animal Carcinogenicity Studies: Implications For The Reach System, Andrew Knight, Jarrod Bailey, Jonathan Balcombe

Jarrod Bailey, PhD

The 2001 European Commission proposal for the Registration, Evaluation and Authorisation of Chemicals (REACH) aims to improve public and environmental health by assessing the toxicity of, and restricting exposure to, potentially toxic chemicals. The greatest benefits are expected to accrue from decreased cancer incidences. Hence the accurate identification of chemical carcinogens must be a top priority for the REACH system. Due to a paucity of human clinical data, the identification of potential human carcinogens has conventionally relied on animal tests. However, our survey of the US Environmental Protection Agency’s (EPA’s) toxic chemicals database revealed that, for a majority of the …

Animal Carcinogenicity Studies: 1. Poor Human Predictivity, Andrew Knight, Jarrod Bailey, Jonathan Balcombe

Jarrod Bailey, PhD

The regulation of human exposure to potentially carcinogenic chemicals constitutes society’s most important use of animal carcinogenicity data. Environmental contaminants of greatest concern within the USA are listed in the Environmental Protection Agency’s (EPA’s) Integrated Risk Information System (IRIS) chemicals database. However, of the 160 IRIS chemicals lacking even limited human exposure data but possessing animal data that had received a human carcinogenicity assessment by 1 January 2004, we found that in most cases (58.1%; 93/160), the EPA considered animal carcinogenicity data inadequate to support a classification of probable human carcinogen or non-carcinogen. For the 128 chemicals with human or …

Which Drugs Cause Cancer?, Andrew Knight, Jarrod Bailey, Jonathan Balcombe

Jarrod Bailey, PhD

Animal tests yield misleading results.

Cancerous Contradictions: The Mis-Regulation Of Human Carcinogens Based On Animal Data, Andrew Knight, Jarrod Bailey, Jonathan Balcombe

Jarrod Bailey, PhD

The regulation of human exposures to potential carcinogens constitutes society’s most important use of animal carcinogenicity data. However, for environmental contaminants of greatest U.S. concern, we found that in most cases (58.1%; 93/160) the U.S. Environmental Protection Agency (EPA) considered the animal data inadequate to support a classification of probable human carcinogen or noncarcinogen.

The World Health Organisation’s International Agency for Research on Cancer (IARC) is a leading international authority on carcinogenicity assessments. For chemicals lacking human exposure data (the great majority), IARC classifications of identical chemicals were significantly more conservative than EPA classifications (p

Which Drugs Cause Cancer?, Andrew Knight, Jarrod Bailey, Jonathan Balcombe

Andrew Knight, Ph.D.

Animal tests yield misleading results.

Cancerous Contradictions: The Mis-Regulation Of Human Carcinogens Based On Animal Data, Andrew Knight, Jarrod Bailey, Jonathan Balcombe

Andrew Knight, PhD

The regulation of human exposures to potential carcinogens constitutes society’s most important use of animal carcinogenicity data. However, for environmental contaminants of greatest U.S. concern, we found that in most cases (58.1%; 93/160) the U.S. Environmental Protection Agency (EPA) considered the animal data inadequate to support a classification of probable human carcinogen or noncarcinogen.

The World Health Organisation’s International Agency for Research on Cancer (IARC) is a leading international authority on carcinogenicity assessments. For chemicals lacking human exposure data (the great majority), IARC classifications of identical chemicals were significantly more conservative than EPA classifications (p

The Future Of Teratology Research Is In Vitro, Jarrod Bailey, Andrew Knight, Jonathan Balcombe

Andrew Knight, PhD

Birth defects induced by maternal exposure to exogenous agents during pregnancy are preventable, if the agents themselves can be identified and avoided. Billions of dollars and manhours have been dedicated to animal-based discovery and characterisation methods over decades. We show here, via a comprehensive systematic review and analysis of this data, that these methods constitute questionable science and pose a hazard to humans. Mean positive and negative predictivities barely exceed 50%; discordance among the species used is substantial; reliable extrapolation from animal data to humans is impossible, and virtually all known human teratogens have so far been identified in spite …

Animal Carcinogenicity Studies: Implications For The Reach System, Andrew Knight, Jarrod Bailey, Jonathan Balcombe

Andrew Knight, PhD

The 2001 European Commission proposal for the Registration, Evaluation and Authorisation of Chemicals (REACH) aims to improve public and environmental health by assessing the toxicity of, and restricting exposure to, potentially toxic chemicals. The greatest benefits are expected to accrue from decreased cancer incidences. Hence the accurate identification of chemical carcinogens must be a top priority for the REACH system. Due to a paucity of human clinical data, the identification of potential human carcinogens has conventionally relied on animal tests. However, our survey of the US Environmental Protection Agency’s (EPA’s) toxic chemicals database revealed that, for a majority of the …

Animal Carcinogenicity Studies: 1. Poor Human Predictivity, Andrew Knight, Jarrod Bailey, Jonathan Balcombe

Andrew Knight, Ph.D.

The regulation of human exposure to potentially carcinogenic chemicals constitutes society’s most important use of animal carcinogenicity data. Environmental contaminants of greatest concern within the USA are listed in the Environmental Protection Agency’s (EPA’s) Integrated Risk Information System (IRIS) chemicals database. However, of the 160 IRIS chemicals lacking even limited human exposure data but possessing animal data that had received a human carcinogenicity assessment by 1 January 2004, we found that in most cases (58.1%; 93/160), the EPA considered animal carcinogenicity data inadequate to support a classification of probable human carcinogen or non-carcinogen. For the 128 chemicals with human or …

Raav2/5 Gene-Targeting To Rods: Dose-Dependent Efficiency And Complications Associated With Different Promoters, William Beltran, Sanford L. Boye, Shannon E. Boye, Vince A. Chiodo, Alfred S. Lewin, William W. Hauswirth, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

A prerequisite for using corrective gene therapy to treat humans with inherited retinal degenerative diseases that primarily affect rods is to develop viral vectors that target specifically this population of photoreceptors. The delivery of a viral vector with photoreceptor tropism coupled with a rod-specific promoter is likely to be the safest and most efficient approach to target expression of the therapeutic gene to rods. Three promoters that included a fragment of the proximal mouse opsin promoter (mOP), the human G-protein-coupled receptor protein kinase 1 promoter (hGRK1), or the cytomegalovirus immediate early enhancer combined with the chicken β actin proximal promoter …

Identification Of Genetic Variation And Haplotype Structure Of The Canine Abca4 Gene For Retinal Disease Association Studies, Barbara Zangerl, Sarah J. Lindauer, Gregory M. Acland, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

Over 200 mutations in the retina specific member of the ATP-binding cassette transporter superfamily (ABCA4) have been associated with a diverse group of human retinal diseases. The disease mechanisms, and genotype–phenotype associations, nonetheless, remain elusive in many cases. As orthologous genes are commonly mutated in canine models of human blinding disorders, canine ABCA4 appears to be an ideal candidate gene to identify and study sequence changes in dogs affected by various forms of inherited retinal degeneration. However, the size of the gene and lack of haplotype assignment significantly limit targeted association and/or linkage approaches. This study assessed the naturally observed …

Modeling The Structural Consequences Of Best1 Missense Mutations, Karina E. Guziewicz, Gustavo D. Aguirre, Barbara Zangerl

Gustavo D. Aguirre, VMD, PhD

Mutations in the bestrophin-1 gene (BEST1) are an important cause of inherited retinal disorders. Hitherto, over 100 unique allelic variants have been linked to the human BEST1 (hBEST1), and associated with disease phenotypes, broadly termed as bestrophinopathies. A spontaneous animal model recapitulating BEST1-related phenotypes, canine multifocal retinopathy (cmr), is caused by mutations in the canine gene ortholog (cBEST1). We have recently characterized molecular consequences of cmr, demonstrating defective protein trafficking as a result of G161D (cmr2) mutation. To further investigate the pathological effects of BEST1 missense mutations, canine and human peptide fragments derived from the protein sequence have been studied …

Identical Mutation In A Novel Retinal Gene Causes Progressive Rod-Cone Degeneration In Dogs And Retinitis Pigmentosa In Humans, Barbara Zangerl, Orly Goldstein, Alisdair R. Philip, Sarah J. P Lindauer, Susan E. Pearce-Kelling, Roberts F. Mullins, Alexander S. Graphodatsky, Daniel Ripoll, Jeanette S. Felix, Edwin M. Stone, Gregory M. Acland, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

Progressive rod–cone degeneration (prcd) is a late-onset, autosomal recessive photoreceptor degeneration of dogs and a homolog for some forms of human retinitis pigmentosa (RP). Previously, the disease-relevant interval was reduced to a 106-kb region on CFA9, and a common phenotype-specific haplotype was identified in all affected dogs from several different breeds and breed varieties. Screening of a canine retinal EST library identified partial cDNAs for novel candidate genes in the disease-relevant interval. The complete cDNA of one of these, PRCD, was cloned in dog, human, and mouse. The gene codes for a 54-amino-acid (aa) protein in dog and human and …

Canine Rd3 Mutation Establishes Rod-Cone Dysplasia Type 2 (Rcd2) As Ortholog Of Human And Murine Rd3, Anna V. Kukekova, Orly Goldstein, Jennifer L. Johnson, Malcolm A. Richardson, Susan E. Pearce-Kelling, Anand Swaroop, James S. Friedman, Gustavo D. Aguirre, Gregory M. Acland

Gustavo D. Aguirre, VMD, PhD

Rod-cone dysplasia type 2 (rcd2) is an autosomal recessive disorder that segregates in collie dogs. Linkage disequilibrium and meiotic linkage mapping were combined to take advantage of population structure within this breed and to fine map rcd2 to a 230-kb candidate region that included the gene C1orf36 responsible for human and murine rd3, and within which all affected dogs were homozygous for one haplotype. In one of three identified canine retinal RD3 splice variants, an insertion was found that cosegregates with rcd2 and is predicted to alter the last 61 codons of the normal open reading frame and further extend …

Comparative Genomic Mapping Of Uncharacterized Canine Retinal Ests To Identify Novel Candidate Genes For Hereditary Retinal Disorders, Barbara Zangerl, Jennifer L. Johnson, Jarek Pillardy, Qi Sun, Catherine André, Francis Galibert, Gregory M. Acland, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

Purpose: To identify the genomic location of previously uncharacterized canine retina-expressed expressed sequence tags (ESTs), and thus identify potential candidate genes for heritable retinal disorders. Methods: A set of over 500 retinal canine ESTs were mapped onto the canine genome using the RHDF5000–2 radiation hybrid (RH) panel, and the resulting map positions were compared to their respective localization in the CanFam2 assembly of the canine genome sequence. Results: Unique map positions could be assigned for 99% of the mapped clones, of which only 29% showed significant homology to known RefSeq sequences. A comparison between RH map and sequence assembly indicated …

Bestrophin Gene Mutations Cause Canine Multifocal Retinopathy: A Novel Animal Model For Best Disease, Karina E. Guziewicz, Barbara Zangerl, Sarah J. Lindauer, Robert F. Mullins, Lynne S. Sandmeyer, Bruce H. Grahn, Edwin M. Stone, Gregory M. Acland, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

PURPOSE. Canine multifocal retinopathy (cmr) is an autosomal recessive disorder of multiple dog breeds. The disease shares a number of clinical and pathologic similarities with Best macular dystrophy (BMD), and cmr is proposed as a new large animal model for Best disease. METHODS. cmr was characterized by ophthalmoscopy and histopathology and compared with BMD-affected patients. BEST1 (alias VMD2), the bestrophin gene causally associated with BMD, was evaluated in the dog. Canine ortholog cDNA sequence was cloned and verified using RPE/choroid 5′- and 3′-RACE. Expression of the canine gene transcripts and protein was analyzed by Northern and Western blotting and immunocytochemistry. …

Development And Validation Of A Canine-Specific Profiling Array To Examine Expression Of Pro-Apoptotic And Pro-Survival Genes In Retinal Degenerative Diseases, Sem Genini, William Beltran, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

We developed an expression profiling array to examine pro-apoptotic and pro-survival genes in dog retinal degeneration models. Gene-specific canine TaqMan assays were developed and included in a custom real-time quantitative reverse transcription-PCR (qRT-PCR) array. Of the 96 selected genes, 93 belonged to known relevant pro-apoptotic and pro-survival pathways, and/or were positive controls expressed in retina, while three were housekeeping genes. Ingenuity Pathway Analysis (IPA) showed that the selected genes belonged to expected biological functions (cell death, cell-mediated immune response, cellular development, function, and maintenance) and pathways (death receptor signaling, apoptosis, TNFR1 signaling, and induction of apoptosis by HIV1). Validation of …

Col9a2 And Col9a3 Mutations In Canine Autosomal Recessive Oculoskeletal Dysplasia, Orly Goldstein, Richard Guyon, Anna Kukekova, Tatyana N. Kuznetsova, Susan E. Pearce-Kelling, Jennifer Johnson, Gustavo D. Aguirre, Gregory M. Acland

Gustavo D. Aguirre, VMD, PhD

Oculoskeletal dysplasia segregates as an autosomal recessive trait in the Labrador retriever and Samoyed canine breeds, in which the causative loci have been termed drd1 and drd2, respectively. Affected dogs exhibit short-limbed dwarfism and severe ocular defects. The disease phenotype resembles human hereditary arthro-ophthalmopathies such as Stickler and Marshall syndromes, although these disorders are usually dominant. Linkage studies mapped drd1 to canine chromosome 24 and drd2 to canine chromosome 15. Positional candidate gene analysis then led to the identification of a 1-base insertional mutation in exon 1 of COL9A3 that cosegregates with drd1 and a 1,267-bp deletion mutation in the …

Steroids Do Not Prevent Photoreceptor Degeneration In The Light-Exposed T4r Rhodopsin Mutant Dog Retina Irrespective Of Ap-1 Inhibition, Danian Gu, William Beltran, Sue Pearce-Kelling, Zexiao Li, Gregory M. Acland, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

PURPOSE. AP-1 has been proposed as a key intermediate linking exposure to light and photoreceptor cell death in rodent light-damage models. Inhibition of AP-1 associated with steroid administration also prevents light damage. In this study the role of steroids in inhibiting AP-1 activation and/or in preventing photoreceptor degeneration was examined in the rhodopsin mutant dog model. METHODS. The dogs were dark adapted overnight, eyes dilated with mydriatics; the right eye was light occluded and the fundus of the left eye photographed (∼15–17 overlapping frames) with a fundus camera. For biochemical studies, the dogs remained in the dark for 1 to …

Linkage Disequilibrium Mapping In Domestic Dog Breeds Narrows The Progressive Rod-Cone Degeneration Interval And Identifies Ancestral Disease-Transmitting Chromosome, Orly Goldstein, Barbara Zangerl, Sue Pearce-Kelling, Duska J. Sidjanin, James W. Kijas, Jeanette Felix, Gregory M. Acland, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

Canine progressive rod–cone degeneration (prcd) is a retinal disease previously mapped to a broad, gene-rich centromeric region of canine chromosome 9. As allelic disorders are present in multiple breeds, we used linkage disequilibrium (LD) to narrow the ∼6.4-Mb interval candidate region. Multiple dog breeds, each representing genetically isolated populations, were typed for SNPs and other polymorphisms identified from BACs. The candidate region was initially localized to a 1.5-Mb zero recombination interval between growth factor receptor-bound protein 2 (GRB2) and SEC14-like 1 (SEC14L). A fine-scale haplotype of the region was developed, which reduced the LD interval to 106 kb and identified …

Long-Term Restoration Of Rod And Cone Vision By Single Dose Raav-Mediated Gene Transfer To The Retina In A Canine Model Of Childhood Blindness, Gregory M. Acland, Gustavo D. Aguirre, Jean Bennett, Tomas S. Aleman, Artur V. Cideciyan, Jeannette Bennicelli, Nadine S. Dejneka, Susan E. Pearce-Kelling, Albert M. Maguire, Krzysztof Palczewski, William W. Hauswirth, Samuel G. Jacobson

Gustavo D. Aguirre, VMD, PhD

The short- and long-term effects of gene therapy using AAV-mediated RPE65 transfer to canine retinal pigment epithelium were investigated in dogs affected with disease caused by RPE65 deficiency. Results with AAV 2/2, 2/1, and 2/5 vector pseudotypes, human or canine RPE65 cDNA, and constitutive or tissue-specific promoters were similar. Subretinally administered vectors restored retinal function in 23 of 26 eyes, but intravitreal injections consistently did not. Photoreceptoral and postreceptoral function in both rod and cone systems improved with therapy. In dogs followed electroretinographically for 3 years, responses remained stable. Biochemical analysis of retinal retinoids indicates that mutant dogs have no …

Genetic And Phenotypic Variations Of Inherited Retinal Diseases In Dogs: The Power Of Within- And Across-Breed Studies, Keiko Miyadera, Gregory M. Acland, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

Considerable clinical and molecular variations have been known in retinal blinding diseases in man and also in dogs. Different forms of retinal diseases occur in specific breed(s) caused by mutations segregating within each isolated breeding population. While molecular studies to find genes and mutations underlying retinal diseases in dogs have benefited largely from the phenotypic and genetic uniformity within a breed, within- and across-breed variations have often played a key role in elucidating the molecular basis. The increasing knowledge of phenotypic, allelic, and genetic heterogeneities in canine retinal degeneration has shown that the overall picture is rather more complicated than …

Congenital Stationary Night Blindness In The Dog: Common Mutation In The Rpe65 Gene Indicates Founder Effect, Gustavo D. Aguirre, Victoria Baldwin, Sue Pearce-Kelling, Kristina Narfström, Kunal Ray, Gregory M. Acland

Gustavo D. Aguirre, VMD, PhD

Purpose: To clone and characterize the canine RPE65 cDNA from normal dog, examine for mutations, and establish if the mutation identified in Swedish briard dogs with retinal dystrophy is present in dogs of the same breed that originated from the United States and other countries, and are affected with congenital stationary night blindness. Methods: Fifteen briard dogs were studied, of which 10 were affected with csnb, and five were clinically normal. In addition, we tested samples from four Swedish dogs, and samples from a briard affected with progressive retinal atrophy. RPE65 cDNA was cloned a from retinal cDNA library by …

Cloning And Characterization Of Canine Pax6 And Evaluation As A Candidate Gene In A Canine Model Of Aniridia, Linda S. Hunter, Duska J. Sidjanin, Manuel Villagrasa Hijar, Jennifer L. Johnson, Ewen Kirkness, Gregory M. Acland, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

Purpose: Mutations in PAX6 cause human aniridia. The small eye (sey) mouse represents an animal model for aniridia. However, no large animal model currently exists. We cloned and characterized canine PAX6, and evaluated PAX6 for causal associations with inherited aniridia in dogs. Methods: Canine PAX6 was cloned from a canine retinal cDNA library using primers designed from human and mouse PAX6 consensus sequences. An RH3000 radiation hybrid panel was used to localize PAX6 within the canine genome. Genomic DNA was extracted from whole blood of dogs with inherited aniridia, and association testing was performed using markers on CFA18. Fourteen PAX6 …

Cloning Of Canine Galactokinase (Galk1) And Evaluation As A Candidate Gene For Hereditary Cataracts In Labrador Retrievers, Duska J. Sidjanin, John L. Mcelwee, Brian Miller, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

We identified a pedigree of Labrador retrievers (LR) that develop hereditary cataracts between 6 and 18 months of age. In humans, galactokinase deficiency is an autosomal recessive disorder characterized by juvenile onset of cataracts.1 In order to evaluate GALK1 as a candidate gene, we cloned and sequenced the canine GALK1 gene and tested a single nucleotide polymorphism (SNP) in the gene for segregation with cataracts in the LR pedigree.

Age-Dependent Disease Expression Determines Remodeling Of The Retinal Mosaic In Carriers Of Rpgr Exon Orfn15 Mutations, William Beltran, Gregory M. Acland, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

PURPOSE. To characterize the retinal histopathology in carriers of X-linked progressive retinal atrophy (XLPRA1 and XLPRA2), two canine models of X-linked retinitis pigmentosa caused, respectively, by a stop and a frameshift mutation in RPGRORF15. METHODS. Retinas of XLPRA2 and XLPRA1 carriers of different ages were processed for morphologic evaluation, TUNEL assay, and immunohistochemistry. Cell-specific markers were used to examine retinal remodeling events. RESULTS. A mosaic pattern composed of patches of diseased and normal retina was first detected in XLPRA2 carriers at 4.9 weeks of age. A peak of photoreceptor cell death led to focal rod loss; however, in these patches …

Blinded By The Light: Retinal Phototoxicity In The Context Of Safety Studies, Maria Cristina De Vera Mudry, Sven Kronenberg, Shun-Ichiro Komatsu, Gustavo D. Aguirre

Gustavo D. Aguirre, VMD, PhD

No abstract provided.

Canine Multifocal Retinopathy In The Australian Shepherd: A Case Report, Ingo Hoffmann, Karina E. Guziewicz, Barbara Zangerl, Gustavo D. Aguirre, Christian Y. Mardin

Gustavo D. Aguirre, VMD, PhD

A 1-year-old Australian Shepherd (AS) was presented for a routine hereditary eye examination. During the examination multiple raised, brown to orange lesions were noted in the fundus, which could not be attributed to a known retinal disease in this breed. As they clinically most closely resembled canine multifocal retinopathy (cmr) and no indication of an acquired condition was found, genetic tests for BEST1 gene mutations were performed. These showed the dog to be homozygous for the cmr1 (C73T/R25X) gene defect. Furthermore, ultrasound (US), electroretinography (ERG), and optical coherence tomography were performed, confirming changes typical for cmr. Subsequently, the AS pedigree …

A Naturally Occurring Mutation Of The Opsin Gene (T4r) In Dogs Affects Glycosylation And Stability Of The G Protein-Coupled Receptor, Li Zhu, Geeng-Fu Jang, Beata Jastrzebska, Slawomir Filipek, Susan E. Pearce-Kelling, Gustavo D. Aguirre, Ronald E. Stenkamp, Gregory M. Acland, Krzysztof Palczewski

Gustavo D. Aguirre, VMD, PhD

Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHO^T4R/T4R dog retina, we found …