Comparative Medicine

Comparative Medicine Volume 57, Number 5, October 2007

OVERVIEW Jones and Cheung. An Introduction to Metabolomics and its Potential Application in Veterinary Science, pp. 436-442

ACLAM Task: Task 3 - Provide Research Support, Information and Services

SUMMARY: This article provides an overview of the application of metabolomics to the area of veterinary science. Metabolomics in this article refers to, "the combination of analytical tools with pattern recognition processes used to define a metabolic phenotype by means of a global approach." More simply put, metabolomics is the analysis of small molecule metabolites that are the product of cellular metabolism.

The analytical methodology used in metabolomics includes the following: nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). NMR offers the advantages of minimal sample preparation, high reproducibility and small sample volumes with the disadvantage of decreased sensitivity when compared to mass spectrometry (MS) based systems. GC-MS systems are useful as they allow quantification of low molecular weight metabolites in gas phase (e.g. from exhaled breath), have increased sensitivity and there are readily searchable databases of fragment patterns. The primary disadvantage of GC-MS is that it can only be used with volatile, thermally stable compounds or those that can be made that way via chemical derivatization thus increasing the processing and analysis time required when compared with other methods. LC-MS methods, like GC-MS are very sensitive but generally require less processing time as volatility of the sample is not required. The major disadvantage of LC-MS methods are that there are numerous ionization techniques in use and fewer spectral libraries available with results from different laboratories varying significantly.

Once samples are processed, the datasets that are generated must be analyzed. Typically this involves the use of multivariate statistics and pattern recognition techniques which can consider the variables in a dataset simultaneously. These multivariate statistical techniques fall into one of two categories: Supervised or unsupervised. Unsupervised techniques model the intrinsic variation found within datasets and do not take class membership (e.g., control vs. diseased animals) into account. Supervised techniques, which include partial least squares-discrimination analysis (PLS-DA), utilize class membership information to maximize separation between groups and better correlate data.

Currently, metabolomics is primarily used in the fields of toxicology and pharmacokinetics as they are used in human pharmaceutical development. In this realm, they are primarily used as an early screening method for toxicity thus removing potentially toxic molecules from the developmental pipeline, minimizing risks and costs. In the veterinary field, the authors state that the use of metabolomics could be applied to veterinary drug development (as it is used in human drug development) as part of the preclinical safety assessment for pharmaceutical compounds. The authors also mention that the noninvasive nature of sample collection (usually urine or plasma are collected) can contribute toward the 3 Rs of reduction, refinement and replacement. In addition to the drug development applications, metabolomics may also have applications in the clinical realm as a diagnostic tool but this has not yet been done on a commercial scale in the human clinical setting and further research would be needed to make this a cost- effective diagnostic method.

The use of metabolomics does have drawbacks with the potential for outside factors such as age, gender, estrus cycle status, to play a significant role in metabolite concentrations which might skew data analysis. In addition diet/nutrition can have a significant impact on the metabolites as can environmental factors such as temperature. Interestingly, the authors mention that these can be accounted for by using additional control animals which contradicts their previous statement about the contribution of metabolomics to the 3Rs.

QUESTIONS: 1. True or False: Metabolomics is widely used in the area of veterinary pharmaceutical development 2. In what 2 areas of human pharmaceutical development is metabolomics widely used? 3. Name 2 methods of metabolite analysis currently in use

ANSWERS 1. False - metabolomics is primarily used in human pharmaceutical development 2. Toxicology and pharmacokinetics 3. NMR, GC-MS, LC-MS (also Fourier transform infrared spectroscopy, Fourier transform ion cyclotron resonance mass spectrometry)

ORIGINAL RESEARCH Mouse Models Haas et al. Effects of Impending Ovarian Failure Induced by 4-Vinylcyclohexene Diepoxide on Fertility in C57BL/6 Female Mice, pp. 443-449

Task 9 - Collaborate on the Selection and Development of Animal Models K1 Animal models (spontaneous and induced) K4 Comparative anatomy, physiology and pathology K5 Characterization of animal models

SUMMARY: The effects of repeated daily dosing with 4-vinylcyclohexene diepoxide (VCD) on the fertility of C57BL/6 female mice on day 16 of pregnancy were characterized. VCD causes a gradual onset of ovarian failure and mice treated with VCD provide a model for perimenopause (the 4 to 10 years that precede menopause) in women. This model is important as an increasing number of women are waiting until they are older to start a family leading to an increased focus on the mechanisms of infertility in aging women. Female C57BL/6 mice were dosed daily for 17 days with vehicle control (sesame oil) or VCD dissolved in sesame oil (160 mg/kg, intraperitoneally) to deplete primordial follicles and divided into 2 groups. Group 1 was mated soon after dosing; group 2 was mated on day 20 after dosing, during impending ovarian failure. VCD-treated mice mated soon (Group 1) after dosing experienced conception difficulties, more resorbed fetuses, and required more matings to become pregnant; however, cycle length, pregnancy rate, and the number of live fetuses did not differ between these mice and controls. Group 2 mice were mated closer to impending ovarian failure and although they demonstrated proestrus and copulatory plugs only one pregnancy was achieved; however, no viable fetuses were present. These results demonstrate that VCD-treated mice can be used to model infertility in perimenopausal women.

Model details: Primordial follicle depletion occurred after 17 days of daily VCD dosing in C57BL/6Hsd female mice. On day 17, all primordial follicles are lost and secondary and antral follicles are unaffected. Ovarian failure occurred an average of 45 days after the onset of 17 days of VCD dosing. Length of initial estrus cycles do not differ between VCD-treated and control animals.

VCD details: Previous rat and mouse studies have shown that VCD (an occupational chemical) specifically targets and destroys primordial and primary follicles while leaving large preantral (secondary) and antral follicles unaffected. The selective follicle loss induced by VCD is due to enhancement of the natural process of atresia which occurs through apoptosis. VCD can be used in mice to accelerate ovarian failure to generate an animal model of peri- and post-menopause. Ovarian failure is not immediate because VCD selectively targets primordial follicles and occurs only after secondary and antral follicles are depleted through ovulation or atresia. The VCD-induced ovarian failure model is more relevant than ovariectomized animals because residual ovarian tissue is retained and the onset of ovarian failure is gradual.

QUESTIONS: 1. What occupational chemical has recently been employed in mice to develop and characterize a mouse model of perimenopausal and postmenopausal ovarian failure relevant to infertility in aging human females? a. Diepoxide cyclohexene b. Diepoxide 4-vinylhexene c. 4-Diepoxide Vinylcyclohexene d. 4-Vinylcyclohexene Diepoxide 2. In the VCD-induced mouse ovarian failure model which follicles are selectively targeted? a. Antral and Primordial b. All follicle types c. Primordial only d. Antral and Preantral e. Primordial and Preantral 3. Which statement accurately reflects the conclusions recently reported in the literature about a VCD-induced mouse model of infertility in perimenopausal women? a. All mice were infertile on the first ovarian cycle following 17 daily doses of VCD b. The cycle length, pregnancy rate, and number of live fetuses did not differ between mice mated during the first two ovarian cycles following 17 daily doses of VCD and controls. c. The cycle length, pregnancy rate, and number of live fetuses did not differ between mice mated 20 days after receiving 17 daily doses of VCD and controls. d. Ovaries from VCD-treated mice contained both follicles and corpora lutea.

ANSWERS: 1. D 2. C 3. B Yang et al. Mice Transgenic for Human Angiotensin-converting Enzyme 2 Provide a Model for SARS Coronavirus Infection, pp. 450-459

SUMMARY: Severe acute respiratory syndrome coronavirus (SARS-CoV) was identified as the etiologic agent of SARS. Nonhuman primates mimic the infection most accurately, but a small animal model would be easier to maintain, more cost effective, and would have less ethical issues. The authors found that the metallopeptidase angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV, indicating that a mouse transgenic for human ACE2 (hACE2) could serve as an animal model for SARS infection. Although, the hACE2 transgenic model described in this article had limited tissue distribution, decreased hACE2 expression, and lack of lethality compared to human SARS; the authors found that it was more susceptible than the wild- type mice, and will still likely facilitate the evaluation of anti-SARS-CoV drugs and vaccines and the analysis of SARS pathogenesis by virus detection and systemic pathologic studies.

QUESTION: 1. List the established animal models for SARS infection:

ANSWER 1. Cynomolgus macaques, ferrets, cats, mice, African green monkeys, and Golden Syrian hamsters.

Seo. Dmrt2 and Pax3 Double-knockout Mice Show Severe Defects in Embryonic Myogenesis, pp. 460-468

ACLAM TASK: Task 9 (animal models) / Task 3 (biomethodology) ACLAM SPECIES: Primary, mouse

SUMMARY: Several transcription factors from the dermomyotome of somites affect embryonic myogenesis. The vast majority of muscles are derived from myotome, and Pax3 and Dmrt2 were studied for their role during embryonic myogenesis. Pax3-/- Dmrt2-/- mice were produced through intercross mating of Pax3+/-Dmrt2-/- mice to C57BL/6J-Pax3SP mice. Mice that were heterozygous for either the Dmrt2 gene or heterozygous for the Pax3 gene were used as wild-type controls because both genes are recessive. The date vaginal plug was observed, was counted as embryonic day 0.5 (E0.5). At E9.5 to E11.5, the critical period for embryonic myogenesis, the embryos were harvested for the following assays:  In situ hybridization was performed on for finding expression levels and patterns of myogenin, Pax3, and Dmrt2,  Immunohistochemistry for desmin, a prominent marker for myocytes throughout myoblast development, was performed on cryosections.  BrdU Immunohistochemistry was performed as a cell proliferation assay. Anti- BrdU was used on fixed, paraffin embedded tissues to determine the amount of BrdU incorporation into embryos (pregnant females were given IP Brd-U, and the E10.5 and E11.5 embryos were harvested 2h post injection of dam, and once they were fixed and embedded).  TUNEL assay was performed on fixed, paraffin embedded tissues. Pax3 and Dmrt2 have similar expression patterns in the dermomyotome of somites. In double-null mutant mice myogenin expression was reduced, and the expression pattern was altered dramatically. Double-null mutants also exhibited phenotype with vertebral and rib malformations, suggesting that the products of both genes have similar or genetically related functions during somitogenesis. In Pax3-knockout mouse embryos, the pattern of Dmrt2 expression was altered, suggesting the Pax3 is important in maintaining the epaxial dermomyotomes.

QUESTIONS: 1. What is TUNEL? a. TUNEL, a structure you drive through on the highway b. TUNEL, a microsurgical technique commonly used in mice and neonates to isolate epithelium/neuroectoderm, called Technique for Ululation of Neuroectodermal or Epithelial Ligation. c. TUNEL, a microsurgical instrument used to isolate muscle fibers or nerves within developing mice. d. TUNEL, Terminal dUTP nick end-labeling, an assay for quantifying apoptosis and DNA fragmentation. 2. What are transcription factors (generally), and which specific transcription factors were studied in this article? 3. Waardenburg syndrome type 3 is characterized by a combination of signs including hypoplasia or contracture of the limb muscles or joints, carpal bone fusion (synostosis), and fusion of digits (syndactyly). With regard to animal model development of human disease, Waardenburg syndrome type 3 is due to homozygous mutation of what gene? 4. What is a somite? 5. What use is the group hypothesizing the Dmrt2-Pax3 mutant mice can be used for?

ANSWERS: 1. d 2. Transcription factors are genes that produce proteins that bind to and regulate the express of DNA. This paper studied dermomyotome transcription factors Pax 3 (paired box gene 3), Dmrt2 (double-sex and mab3 related factor 2), and myogenin. 3. PAX 3 mutation leads to Waardenburg syndrome type 3. 4. The somites are paired embryonic structures derived from presomitic mesoderm during vertebral development. It differentiates into dermomyotome and sclerotome. The dermomyotome develops into dermatome and myotome. The sclerotome ultimately becomes the bones of the body axis. 5. To elucidate role in innate muscular dystrophy and provide model for drug development to promote muscle repair after injury.

Li et al. Phenotypic Analysis of C57BL/6J and FVB/NJ Mice Generated Using Evaporatively Dried Spermatozoa, pp. 469-475

Primary Species: Mus musculus Task 4 Develop And Manage Animal Husbandry Programs

SUMMARY: Cryopreservation of mouse sperm using liquid nitrogen is a routine procedure to preserve sperm for in vitro fertilization or artificial insemination. However; sperm from strains of mice like C57BL/6J and FVB/NJ suffer variable degrees of cryodamage following liquid nitrogen cryopreservation. A recent technique developed as a breeding strategy is to inject spermatozoan into the cytoplasm of ovum in a process referred to as intracytoplasmic sperm injection (ICSI). ISCI allows for sperm to be preserved in alternate ways such as freeze drying and evaporative drying. Advantages of evaporative drying include; does not require liquid nitrogen or intricate machinery, making it a simpler, faster and a cheaper method compared to freeze drying. The purpose of this study was to demonstrate the viability of evaporatively dried sperm from C57Bl/6J and FVB/NJ mice following 30 days of freezing at -80C.

In the ICSI procedure fertilized 1-cell stage eggs were cultured to the 2-cell stage and the embryos were then transferred into the oviducts of pseudopregnant CD1 recipient mice .5 day postcoitus with a vasectomized stud male.

Evaporative drying and frozen storage did not have deleterious effects on fertilization rates and embryo development compared to fresh sperm for either strain. The body weights of the first generation pups were also found to have no differences compared to pups generated by fresh sperm. It was again observed that there were no differences in litter size, gender ratio, and body weight in second generation mice. There were no increased susceptibility to adventitious pathogens or unusual pathological changes observed through post mortem and histology.

QUESTIONS: 1. What is ICSI short for? 2. Give three advantages of evaporative drying over liquid nitrogen cryopreservation. 3. T/F Evaporative drying has deleterious effects on embryo development compared to cryopreservation

ANSWERS: 1. Intracytoplasmic sperm injection 2. Advantages of evaporative drying include; does not require liquid nitrogen or intricate machinery, making it a simpler, faster and a cheaper method compared to freeze drying. 3. F

Mic et al. Indomethacin Inhibits Thymic Involution in Mice with Streptozocin- induced Diabetes, pp. 476-481

ACLAM TASK: Task 9 ACLAM SPECIES: Primary, mouse

SUMMARY: Streptozotocin (STZ)-induced hyperglycemia in mice is an experimental model of diabetes that leads to persistent hyperglycemia and complications that mimic some of the manifestations of diabetes in humans. Upon induction of diabetes by STZ, the thymus involutes quickly, although the mechanism is unclear. Some studies have shown that STZ is toxic to primary lymphocytes, causing thymocyte apoptosis. It has also been shown that chronic hyperglycemia activates the stress response that is associated with elevated glucocorticoids, which are known inducers of apoptosis in thymocytes. Recent studies show mild inflammation accompanies type 2 diabetes and that inhibition of cyclooxygenase 2 prevents hyperglycemia in a STZ-induced diabetic mouse model. Because cyclooxygenase 2 and arachidonic acid metabolites are involved in glucocorticoid-induced thymic involution, this study investigated the role of the cyclooxygenase 2 signaling pathway in diabetes-induced thymic involution.

Four to five week old NMRI mice were injected with STZ alone or STZ + indomethacin (INDO), and euthanized at various time points for thymus harvest. INDO is a known cyclooxygenase 2 inhibitor. Thymus weight, glycemia, and serum corticosterone were measured, and apoptosis in thymus and thymocytes cultures were measured by flow cytometry. Consistent with previous data, a single massive dose of STZ led to rapid hyperglycemia, thymic involution, and weight loss. Surprisingly, however, even massive doses of STZ did not induce thymocyte apoptosis. Thymocytes were also unaffected by high glucose concentrations, even after 24 hrs of exposure in culture. Administration of INDO concurrently with STZ inhibited thymic involution but did influence hyperglycemia. When INDO was given 4 hours before STZ, thymic involution was reduced to the same degree as when INDO was given simultaneously with STZ, but hyperglycemia was reduced. INDO also reduced serum corticosterone.

In this study, corticosterone (the main glucocorticoid in mice) began to increase after induction of diabetes. This is consistent with the proinflammatory and stress-inducing activity of hyperglycemia. The authors suggest that elevated corticosterone might offer an explanation for the rapid induction of the thymus in experimental diabetes, given that thymocytes are sensitive to glucocorticoids. The inhibition of cyclooxygenase 2 by INDO may lead to systemic reduction of corticosterone levels, potentially explaining the reduction in thymic involution. They conclude that thymic involution is unlikely to be caused by STZ or hyperglycemia, and that the cyclooxygenase 2 pathway is involved in thymic involution during diabetes.

QUESTIONS: 1. Streptozotocin administration is primarily used to create an animal model of which condition? a. Glomeruloneohritis b. Cholangiohepatitis c. Diabetes d. Myocardial fibrosis 2. The authors suggest that which of the following is responsible for thymic involution during diabetes? a. Hyperglycemia b. Hypoglycemia c. Streptozotocin toxicity d. Cyclooxygenase 2 mediated mechanisms 3. What difference was seen between mice administered INDO concurrently with STZ (group A) and mice administered INDO before STZ (group B)? a. Reduced thymic involution in Group A as compared to Group B b. The same degree of thymic involution in both groups, but reduced hyperglycemia in Group B c. Reduced thymic involution and reduced hyperglycemia in Group B d. No difference

ANSWERS: 1. c 2. d 3. b

Shao et al. Exposure of Preimplantation Embryos to Insulin Alters Expression of Imprinted Genes, pp. 482-486

SUMMARY: This study measures whether exposure of pre-implantation mouse embryos to insulin would affect fetal development and genetic expression. Previously, insulin has been shown to affect a number of measures of fetal development, including protein synthesis, and blastocyst formation. In addition, several genes (H19 and Igf2) have also been shown to influence fetal development including birth weight. However, links between insulin and these genes have not previously been identified.

Allelic methylation status is important to the expression of both H19 and Igf2 genes. Increased allelic methylation, or the addition of a methyl group to a cytosine residue, could result in decreased gene expression. The methylation status of H19 and Igf2 can be affected by nutritional status of the mother, as well as exposure to environmental toxins and radiation. The authors speculate that if insulin is acting at the gene expression level during embryonic development allelic methylation status could be a mechanism.

Super-ovulated female mice were allowed to mate and embryos were collected from the successfully impregnated females at the two-cell stage. Embryos were divided equally into either a control group or an insulin-exposed group. All embryos were cultured in KSOM medium for 48 hours; however the medium in the insulin-exposed group was supplemented with insulin. The embryos were implanted and allowed to gestate for 14 days. The number of blastocysts at 48-hours and weight of the fetus following 14-day gestation indicated that insulin-exposed embryos developed quicker and were larger than controls. Whole body gene expression levels were higher for both H19 and Igf2 in the insulin-exposed fetuses. Finally, nucleotide sequences of insulin-exposed fetuses indicated decreased rates of allelic methylation.

The authors report that insulin stimulates embryonic development by altering the expression levels of H19 and Igf2 through the methylation status of those genes. The authors speculate that this study provides insights into probable mechanisms for early disruptions in embryonic development due to environmental or nutritional factors.

QUESTIONS 1. T or F? Insulin accelerated blastocyst formation and enhanced birth weight of mouse fetuses by 17.8% compared to controls. 2. Over expression of which genes can cause fetal overgrowth? a. IgG and IgA b. Igf2 and H19 c. A and B d. H20 and RNA 3. Which factors influence the genomic DNA methylation status of the imprint control region of H19/Igf2 in vitro? a. Nutritional factors b. Radiation c. Toxic environmental contaminant d. Culture medium e. All of the above 4. T or F? Insulin increased rates of allelic methylation and increased gene expression.

ANSWERS: 1. True 2. B 3. E 4. False

Rat Model Hunter et al. Fenbendazole Treatment May Influence Lipopolysaccharide Effects in Rat Brain, pp. 487-492

SUMMARY: Pinworms are common parasites in rodent colonies and are commonly treated with Fenbendazole, a benzimidazole anthelminthic which has adulticidal, larvicidal and ovicidal activity. Benzimadazoles act to interfere with nematode microtubule assembly and disassembly by interacting with β-tubulin. This effect can also interfere with mammalian cellular functions and is thought to have sufficient bioavailability to interact with other drugs or toxins in the brain. This retrospective study was undertaken to explain previously noted effects of combined treatment of rats with lipopolysaccharide (LPS) and fenbendazole (FBZ). The authors used F344 rats that were treated with intrastriatal injections of LPS or saline and compared the effects of animals receiving fenbendazole treatment and without. Parameters for analysis were weight loss, microglial activation in the substatia nigra, astrocyte loss in the substantia nigra, and striatal dopamine.

The authors found that rats that received fenbendazole after LPS administration had weight loss (approx. 5%) and greater microglial activation. This treatment combination was also associated with reduced striatal dopamine levels.

Prolonged microglial activation supports the view that fenbendazole has an immunomodulating effect which is in contrast to earlier reports that the drug does not alter the immune response. The reduction of striatal dopamine levels implies that the combined LPS and FBZ damages dopaminergic neurons to a greater degree that LPS alone. This leads the authors to speculate that FBZ promotes LPS induced microglial response to cause neurotoxicity and/or FBZ modulates the dopaminergic system. LPS injection alone decreased the number of GFAP-positive astrocytes and FBZ treatment mitigated this decrease indicating that FBZ may have a protective effect against LPS toxicity to astrocytes. The mechanism for this effect is unknown.

The authors point out that this study was not repeated and that control group and experimental group were not studied at the same time but other factors were controlled.

QUESTIONS: 1. Fenbendazole when used as a treatment for Pinworms has immunomodulating effect in F344 rats. T or F? 2. Fenbendazole treatment when combined with intrastriatal injections of LPS has all of the following effects except: a) Increased microglial activity in the substatia nigra b) Weight loss of approximately 5% c) Further decreased the number of astrocytes in the substantia nigra d) Reduced striatal dopamine levels 3. Fenbendazole has larvicidal, and ovicidal effects on pinworms but does not affect adults. T or F?

ANSWERS: 1. T 2. C 3. F

Pig Model Christoffersen et al. Gender-associated Differences in Metabolic Syndrome- related Parameters in Gottingen Minipigs, pp. 493-504

Task Designation: 1K2: Prevent, Diagnose, Control, and Treat Disease, Physiology Species Designation: Primary

SUMMARY: This study characterized gender differences in metabolic parameters of Gottingen minipigs to determine which gender has the metabolic profile that is most appropriate as a model for human metabolic syndrome. Experimental groups consisted of 8 week old and 8 month old male and female minipigs. The authors measured glucose, fructosamine, insulin, C-peptide, glucagon, triglycerides, total cholesterol, HDL- c, free fatty acids, leptin, testosterone, and 17B-estradiol. Insulin sensitivity and beta- cell function testes were also performed. Male minipigs had higher concentrations of both testosterone and estradiol. Female minipigs had higher concentrations of C- peptide, insulin, triglycerides, total cholesterol, HDL-c, and leptin. Female pigs were also more insulin-resistant and had higher beta-cell function than the males. The authors concluded that the female minipigs may be a better model for human metabolic syndrome than male minipigs.

QUESTIONS: 1. T/F There are minimal physiologic differences between different genders of Gottingen minipigs. 2. T/F Minipigs are a poor model for human metabolic syndrome. 3. T/F Sex hormones can influence obesity and body condition scoring.

ANSWERS: 1. F 2. F 3. T

Nonhuman Primate Model Mills et al. Ingestion of Excessive Preformed Vitamin A by Mothers Amplifies Storage of Retinyl Esters in Early Fetal Livers of Captive Old World Monkeys, pp. 505-511 Task 1, 3, 4 Primary species: Macaca mulatta, Macaca facicularis, Tertiary species: Chlorocebus aethiops This study measures liver concentrations of vitamin A (VA) in early and midgestation old world fetal monkeys. Animals included 11 M. mulatta, 4 M. facicularis, and 4 C. aethiops. These monkeys are considered comparable to humans due to fetal organ growth rates and sizes, as well as similarities in reproductive cycle, gestational length, placental structure, and postnatal development.

Retinyl palmitate is the major dietary and storage form of VA. Retinyl palmitate is metabolized to retinol in the GI and liver, and further oxidized to retinal (for vision) and retinoic acid (mediates gene transcription for tissue growth and differentiation). Trans, 13 cis and 13 cis 4 oxo retinoic acid are teratogenic. In mice, prior to increased hepatic maturity for metabolism of VA at midgestation, embryos rely on placental transport of retinoic acid. VA restriction impairs growth, prolongs gestation, increases fetal resorption, and can result in teratogenesis. VA excess in the diet results in congenital malformation of various sorts.

Commercial primate laboratory diet daily intake has been linked to elevated liver and serum retinyl esters, stellate cell hypertrophy and hyperplasia, and extrahepatic VA storage in adult monkeys, suggestive of VA toxicity. In rats and humans, liver VA stores increase during mid to late gestation. Early to mid gestational liver stores have not been examined in old world monkeys previously.

Fetal livers were obtained from animals delivered by caesarian from adult lifetime captive mothers. These were obtained after embryos were used for other studies. Mothers were fed Teklan Global Primate Diet 2050, with 22.58 IU retinyl acetate/g, at a rate within the 2-4% bodyweight recommended levels. (Levels of VA obtained from dietary enrichment were not considered.) HPLC was used to establish levels of retinol, retinyl esters, and retinoic acid from fetal livers. The data revealed that there was a greater rate of VA accumulation in midgestation than early gestation, and that levels were much higher than in other mammals examined. Retinyl esters were the predominant form of VA in the liver during early and middle gestation. Retinol was higher in early gestation than middle. The authors conclude, from comparisons between published fetal liver VA values in humans and late gestational age rhesus, that the levels reported in this study represented elevated retinyl ester concentration, and are indicative of hypervitamintosis A in mothers, though this was not linked to congenital defects. The authors speculate that maternal VA is preferentially shunted to retinyl esters for storage as a protective mechanism in the fetus.

QUESTIONS: 1. All trans and all cis retinoic acids are teratogens. T/F 2. What was the predominant form of VA stored in the liver in both early and mid gestation? 3. What are the signs of hypervitamintosis A in mature animals? 4. Are normal fetal liver stores of VA known from old world monkeys?

ANSWERS: 1. False 2. Retinyl esters 3. Elevated liver and serum retinyl esters, stellate cell hypertrophy and hyperplasia, and extrahepatic VA storage 4. No – all conclusions in this paper result from extrapolation in other species.