The guinea pig as an animal model for developmental and reproductive toxicology studies

BACKGROUND : Regulatory guidelines for developmental and reproductive toxicology (DART) studies require selection of “relevant” animal models as determined by kinetic, pharmacological, and toxicological data. Traditionally, rats, mice, and rabbits are the preferred animal models for these studies. However, for test articles that are pharmacologically inactive in the traditional animal models, the guinea pig may be a viable option. This choice should not be made lightly, as guinea pigs have many disadvantages compared to the traditional species, including limited historical control data, variability in pregnancy rates, small and variable litter size, long gestation, relative maturity at birth, and difficulty in dosing and breeding. METHODS : This report describes methods for using guinea pigs in DART studies and provides results of positive and negative controls. Standard study designs and animal husbandry methods were modified to allow mating on the postpartum estrus in fertility studies and were used for producing cohorts of pregnant females for developmental studies. RESULTS : A positive control study with the pregnancy-disrupting agent mifepristone resulted in the anticipated failure of embryo implantation and supported the use of the guinea pig model. Control data for reproductive endpoints collected from 5 studies are presented. CONCLUSION : In cases where the traditional animal models are not relevant, the guinea pig can be used successfully for DART studies. Birth Defects Res (Part B)86: 92-97, 2009. © 2009 Wiley-Liss, Inc.     

An Evaluation of Reproductive and Developmental Toxicity of Sitaxentan (Thelin) in Rats

Sitaxentan sodium (Thelin) is a once daily, orally bioavailable, highly selective endothelin A receptor antagonist. Initially approved for the treatment of pulmonary arterial hypertension, sitaxentan was withdrawn in 2010 following the recognition of a pattern of idiosyncratic liver injury. During development of this drug, a series of nonclinical studies investigated the effects of orally administered sitaxentan on fertility, embryofetal development, and pre‐ and postnatal development in the rat; results of these studies are reported here. In the fertility study, sitaxentan did not affect mating behavior, fertility, sperm morphology, or estrous cycle. Sitaxentan was teratogenic in the embyrofetal development study, which was expected based on its pharmacologic mechanism of action. Teratogenic effects included malformations of the head, mouth, face, and large blood vessels. In the pre‐ and postnatal study, sitaxentan administration was associated with reduced pup survival, large or abnormally shaped livers, and delays in markers of auditory and sexual development. Sitaxentan was detected in plasma of suckling pups receiving milk from females dosed with sitaxentan. These nonclinical study findings were reflected in the sitaxentan product label warnings. Birth Defects Res (Part B) 00:1‐10, 2012. © 2012 Wiley Periodicals, Inc.     

Wound-healing studies in transgenic and knockout mice

Injury to the skin initiates a cascade of events including inflammation, new tissue formation, and tissue remodeling, that finally lead to at least partial reconstruction of the original tissue. Historically, animal models of repair have taught us much about how this repair process is orchestrated and, over recent years, the use of genetically modified mice has helped define the roles of many key molecules. Aside from conventional knockout technology, many ingenious approaches have been adopted, allowing researchers to circumvent such problems as embryonic lethality, or to affect gene function in a tissue-or temporal-specific manner. Together, these studies provide us with a growing source of information describing, to date, the in vivo function of nearly 100 proteins in the context of wound repair. This article focuses on the studies in which genetically modified mouse models have helped elucidate the roles that many soluble mediators play during wound repair, encompassing the fibroblast growth factor (FGF) and transforming growth factor-β (TGF-β) families and also data on cytokines and chemokines. Finally, we include a table summarizing all of the currently published data in this rapidly growing field. For a regularly updated web archive of studies, we have constructed a Compendium of Published Wound Healing Studies on Genetically Modified Mice which is available at http://icbxs.ethz.ch/members/grose/woundtransgenic/home.html.     

Developmental and reproductive toxicity studies on artemisone

BACKGROUND: In order to justify clinical studies in women of child-bearing age with artemisone, a new artimisinin derivative, studies to assess fertility and early embryonic development in rats, developmental toxicity in rats and rabbits, and peri-post natal development in rats were performed. METHODS AND RESULTS: In the study on fertility and early embryonic development (dose levels 0-5-20-80 mg/kg bw/day), doses inducing clinical and organ toxicity were used. Only in severe toxicity conditions, a reduction of the number of estruses, a prolonged time to insemination, decreased numbers of corpora lutea, implantation sites, and viable fetuses were found. Two developmental toxicity studies were performed in rats (dose levels 0-1-2 mg/kg bw/day) and rabbits (dose levels 0-2.5-5.0-7.5 mg/kg bw/day). It was shown that rats were about 5 times more sensitive than rabbits. In rats, artemisone induced total litter loss (late resorptions) at 2 mg/kg body weight and above with an increased incidence of a common vascular variation and retarded ossification at this dose. In rabbits, maternal toxicity, abortion and a slightly increased incidence of cardiac ventricular septal defects was observed at 7.5 mg/kg body weight. In a pre- and postnatal developmental toxicity study in rats (dose levels 0-1-2-4 mg/kg bw/day), 4 mg/kg body weight artemisone induced clinical symptoms and affected postnatal survival, body weight gain in the F1 pups, and motor activity. CONCLUSIONS: In summary, artemisone was shown to be embryo- and fetotoxic and induced cardiac ventricular septal defects and retarded ossification in dosages where total litter loss and abortions were observed. However, no effect on reproductive and developmental parameters below severe toxic dosages could be observed. Birth Defects Res (Part B)86: 131-143, 2009. © 2009 Wiley-Liss, Inc.     

Fertility and Developmental Toxicity Assessment in Rats and Rabbits with LY500307, a Selective Estrogen Receptor Beta (ERβ) Agonist

LY500307 is a selective estrogen receptor beta (ERβ) agonist that was developed for the treatment of benign prostatic hyperplasia. The in vitro functional selectivity of LY500307 for ERβ agonist activity is 32‐fold above the activity at the alpha receptor (ERα). LY500307 was evaluated in a series of male (M) and female (F) rat fertility and rat and rabbit embryo‐fetal development (EFD) studies, using 20 or 25 animals/group. LY500307 was administered daily by oral gavage starting 2 weeks (F) or 10 weeks (M) before mating, during cohabitation, until necropsy (M) or through gestation day (GD) 6 (F) in the fertility studies and from GD 6 to 17 (rats) or GD 7 to 19 (rabbits) in the EFD studies. Dosage levels of LY500307 ranged from 0.03 to 10 mg/kg/day for rats and from 1 to 25 mg/kg/day for rabbits. Fertility, estrous, maternal reproductive endpoints, conceptus viability, sperm parameters, organ weights, and histopathology were evaluated in the fertility studies. Maternal reproductive endpoints and fetal viability, weight, and morphology were evaluated in the EFD studies. Toxicokinetics were assessed in satellite animals. At 10 mg/kg/day in the male fertility study, findings included decreased body weight (BW); food consumption (FC); fertility, mating, and conception indices; sperm concentration; and reproductive tissue weight (associated with atrophic histologic changes). In the female fertility study, effects included decreased BW and FC at ≥0.3 mg/kg/day and persistent diestrus, delayed mating, and reduced fertility/conception indices at 3 mg/kg/day. In the rat EFD study, findings included decreased maternal BW and FC and increased incidences of adverse clinical signs, abortion, maternal mortality/moribundity, postimplantation loss, and fetal skeletal variations at 3 mg/kg/day. Effects in the rabbit EFD study were limited to decreases in maternal BW and FC at 25 mg/kg/day. In general, systemic maternal exposure increased proportionally with dosage in rats, but less than proportionally in rabbits. In conclusion, the no‐observed adverse effect levels following LY500307 administration were 1 mg/kg/day for male rat fertility, 0.3 mg/kg/day for female rat fertility and EFD, and 25 mg/kg/day for rabbit EFD. Adverse reproductive and developmental effects only occurred at or above parentally toxic dosage levels and were considered predominantly due to off‐target ERα effects.     

Developmental potential of isolated blastomeres from early murine embryos

Experiments were designed to evaluate the effect of blastomere separation on blastocoele formation and development of viable fetuses. Two-cell and four-cell murine embryos were dissociated into individual blastomeres and cultured to the blastocyst stage. For embryos of both stages, zona removal and blastomere separation reduced (P<0.05) the number of viable embryos at the onset of culture and reduced (P<0.01) the frequency of continuation of development of blastomeres to the blastocyst stage. Attempts to repeatedly split two-cell stage embryos decreased in vitro development to blastocysts. The number of cells in two-cell embryos that were cultured to blastocyst was not different for control (64.8 +/- 11.5) or for two-cell embryos cultured without the zona pellucida (60.9 +/- 10.1) but was reduced (P<0.01) for one-half embryos that were cultured to blastocysts (35.6 +/- 10.6). The cell number of blastocysts obtained from dissociated four-cell (1/4) embryos (17.4 +/- 1.4) was similarly reduced (P<0.01). In vivo development was assessed after cultured embryos were transferred to the uteri of day 3 pseudopregnant females. Zona free intact embryos (2/36, 6%) and zona free half embryos (7/36; 19%) developed less frequently (P<0.05) than intact controls (45/100). Noncultured morula briefly exposed to pronase to thin the zona had similar impaired development. Embryos with thinned zona or no zona developed less frequently (21/82, 2/72 respectively, P<0.05) than nonpronase-treated controls (50/83).     

Interferon-gamma production and host protective response against Mycobacterium tuberculosis in mice lacking both IL-12p40 and IL-18

Interferon (IFN)-gamma plays an essential role in host defense against infection with Mycobacterium tuberculosis, and its synthesis is critically regulated by interleukin (IL)-12, IL-18 and the recently identified IL-23. The present study was designed to determine the roles of these cytokines in IFN-gamma-mediated host defenses against M. tuberculosis. For this purpose, we compared host protective responses in IL-12p40 and IL-18 double-knockout (DKO) mice (which lacked both IL-12/IL-18 and also IL-23) and IFN-gamma gene-disrupted (GKO) mice. DKO mice were more resistant to the infection than GKO mice, as indicated by their extended survival and reduced live colony numbers in spleen, liver and lung. IFN-gamma was detected by ELISA in liver and lung homogenates, but not in spleen and serum, and in all organs by RT-PCR in DKO mice at comparable or reduced levels to those in wild-type mice. IFN-gamma production was reduced by depletion of CD4+ T cells, but not of natural killer (NK), NKT, gammadeltaT and dendritic cells. Neutralization of IFN-gamma or TNF-alpha by specific monoclonal antibodies (mAbs) significantly shortened the survival time of the infected DKO mice. Furthermore, anti-TNF-alpha mAb partially attenuated IFN-gamma synthesis in the liver of these mice. Finally, the expression level of inducible nitric oxide synthase (iNOS) mRNA in the spleen, liver and lung was considerable in DKO mice but only marginal or undetected in GKO mice. Our results indicate the presence of IL-12-, IL-18- and IL-23-independent host protective responses against mycobacterial infection mediated by IFN-gamma, which was secreted from helper T cells.     

Reproduction in mice: Spermatozoa as factors in the development and implantation of embryos

The effects on mouse embryo development in vivo of varying the numbers of spermatozoa used in artificial inseminations was studied. The two criteria used in the evaluation of the progress of embryo development were 1) ability to reach the two-cell stage and 2) success of development from the two-cell stage through implantation. A 44% reduction in the yield of two-cell embryos and a 67% reduction in the number of implants was observed when C3HeB/FeJ females were inseminated with one-twentieth the number of spermatozoa estimated to be present in a typical ejaculate. The reduction in the yield of two-cell embryos was substantially reversed by a second insemination of a large number of heat-inactivated spermatozoa 12 hr after the first insemination. The sperm-dependent reduction in development from the two-cell stage through implantation was prevented only by normal viable (unheated) spermatozoa. These results were rationalized by the hypothesis that in female C3HeB/FeJ mice spermatozoa serve physiological functions beyond the fertilization of ova and that spermatozoa may act to foster early embryo development through modulation of the environments embryos experience as they move through the reproductive tract.     

Developmental toxicity research of ginsenoside Rb1 using a whole mouse embryo culture model

BACKGROUND: Ginseng has been widely used around the world for many years. Knowledge is limited, however, on its effects on embryonic development. METHODS: Whole embryo culture was used to explore the developmental toxicity of ginsenoside Rb1 (GRb1) on mouse embryos. All embryos were exposed to different concentrations of GRb1, and scored for their growth and differentiation at the end of the 48-hr culture period. RESULTS: Total morphological score decreased significantly at the concentration of GRb1 of 30 microg/ml and was further reduced at 50 microg/ml. Yolk sac was affected at the lower concentration of 15 microg/ml. Developments of midbrain, forebrain, and optic system were relatively sensitive to GRb1 and were affected at the concentration of 30 microg/ml. Allantois, flexion, branchial arch, and limb buds were affected at 50 microg/ml. At this concentration, the embryonic crown-rump length, head length, and somite number were also reduced significantly compared to the control group. CONCLUSIONS: These results suggest that GRb1 has teratogenic effect during the mouse organogenetic period. We suggest that before more data in humans is available, ginseng should be used with caution by pregnant women in the first trimester.     

Chlamydia trachomatis mouse pneumonitis lung infection in IL-18 and IL-12 knockout mice: IL-12 is dominant over IL-18 for protective immunity

BACKGROUND: Interferon (IFN)-gamma is a key to protective immunity against a variety of intracellular bacterial infections, including Chlamydia trachomatis. Interleukin (IL)-18, a recently identified Th1 cytokine, together with IL-12 is a strong stimulator for IFN-gamma production. We investigated the relative roles of IL-18 and IL- 12 in protective immunity to C. trachomatis mouse pneumonitis (MoPn) infection using gene knockout (KO) and wild-type (WT) mice. MATERIALS AND METHODS: Mice were intranasally infected with C. trachomatis MoPn and protective immunity was assessed among groups of mice by daily body weight changes, lung growth of MoPn, and histopathological appearances at day 10 postinfection. The corresponding immune responses for each group of mice at the same postinfection time point were evaluated by measuring antigen-specific antibody isotype responses and cytokine profiles. RESULTS: Our results showed that IL-18 deficiency had little or no influence on clearance of MoPn from the lung, although KO mice exhibited slightly more severe inflammatory reactions in lung tissues, as well as reduced systemic and local IFN-gamma production, compared with WT mice. Results with IL-18 KO mice were in sharp contrast to those observed with IL-12 KO mice that showed substantially reduced clearance of MoPn from the lungs, substantial reductions of antigen-specific systemic and lung IFN-gamma production, decreased ratio of MoPn-specific immunoglobulin G (IgG)2a/IgG1, and severe pathological changes in the lung with extensive polymorphonuclear, instead of mononuclear, cell infiltration. Exogenous IL-12 or IL-18 was able to increase IFN-gamma production in IL-18 KO mice; whereas, only exogenous IL-12, but not IL-18, enhanced IFN-gamma production in IL-12 KO mice. Caspase-1 is the key protease for activation of IL-18 precursor into the bioactive form, and caspase-1 KO mice also displayed similar bacterial clearance and body weight loss to that in WT mice at early stages of MoPn infection. This further confirmed that IL-18 was not essential for host defense against chlamydia infection. CONCLUSIONS: These results suggest that IL-12, rather than IL-18, plays the dominant role in the development of protective immunity against chlamydia lung infection, although both cytokines are involved in the in vivo regulation of IFN-gamma production.     

Dendritic cell-derived IL-12p40 homodimer contributes to susceptibility in cutaneous leishmaniasis in BALB/c mice

Protection against Leishmania major in resistant C57BL/6 mice is mediated by Th1 cells, whereas susceptibility in BALB/c mice is the result of Th2 development. IL-12 release by L. major-infected dendritic cells (DC) is critically involved in differentiation of Th1 cells. Previously, we reported that strain differences in the production of DC-derived factors, e.g., IL-1alphabeta, are in part responsible for disparate disease outcome. In the present study, we analyzed the release of IL-12 from DC in more detail. Stimulated DC from C57BL/6 and BALB/c mice released comparable amounts of IL-12p40 and p70. In the absence of IL-4, BALB/c DC produced significantly more IL-12p40 than C57BL/6 DC. Detailed analyses by Western blot and ELISA revealed that one-tenth of IL-12p40 detected in DC supernatants was released as the IL-12 antagonist IL-12p40 homodimer (IL-12p80). BALB/c DC released approximately 2-fold more IL-12p80 than C57BL/6 DC both in vitro and in vivo. Local injection of IL-12p80 during the first 3 days after infection resulted in increased lesion volumes for several weeks in both L. major-infected BALB/c or C57BL/6 mice, in higher lesional parasite burdens, and decreased Th1-cytokine production. Finally, IL-12p40-transgenic C57BL/6 mice characterized by overexpression of p40 showed increased levels of serum IL-12p80 and enhanced disease susceptibility. Thus, in addition to IL-1alphabeta, strain-dependent differences in the release of other DC-derived factors such as IL-12p80 may influence genetically determined disease outcome.     

Exogenous interleukin-12 (IL-12) exacerbates Cryptosporidium parvum infection in gamma interferon knockout mice

Experimental infection of BALB/c- or C57BL/6-gamma-interferon-knockout (GKO) mice with Cryptosporidium parvum results in infection in both strains with different outcomes of disease. The BALB/c-GKO mice recover from infection, whereas the C57BL/6-GKO mice succumb to infection in less than 2 weeks. Differences in cytokine mRNA expression suggested that recovery may involve other cytokines. To determine whether the addition of either a Th1 or Th2 cytokine could alter the outcome of infection, we treated GKO mice with either recombinant (r)IL-4 or rIL-12 1 day before infection (DBI) or daily. No effect on the oocyst shedding patterns in either strain nor an increase in survival of the C57BL/6-GKO mice was observed in the rIL-4-treated mice. Whereas one dose of 0.5 microg rIL-12 given 1 DBI had no effect on oocyst shedding, we found that daily doses of rIL-12 administered intraperitoneally exacerbated C. parvum infection in both animal models. Administration of rIL-12 shortened the survival time in the C57BL/6-GKO mice and prevented BALB/c-GKO mice from recovering from infection. Specific proliferation of T cells to cryptosporidial antigen and Th1 and Th2 mRNA cytokine expression was markedly decreased in rIL-12-treated mice. Nitric oxide (NO) may have played a minor role in the decreased proliferation observed since levels of NO present in the splenocyte cultures from rIL-12-treated mice in response to parasite antigen stimulation were higher than those observed in controls. Thus, we propose that resistance to and recovery from C. parvum infections involves a fine balance in the amount and timing of Th1 and Th2 cytokines.     

Experimental studies on reproductive toxicologic effects of lamotrigine in mice

BACKGROUND: Virtually all antiepileptic drugs (AED) tested so far have been found to be teratogenic. The second generation AED possess a number of therapeutic advantages over the older ones. There are, however, very little data on their effects on embryonic development. A recent report suggests that lamotrigine (LTG) can be teratogenic to human fetuses. With only a few cases of prenatal exposure to LTG in the record, however, it has not been possible to establish a recognizable pattern of malformations in the infants of LTG‐treated mothers. OBJECTIVES: The objectives of the present study were to evaluate the reproductive toxic effects of LTG RESULTS: Single (50–200 mg/kg) or multiple doses (25, 50, 75 mg/kg) of LTG were administered by intraperitoneal (i.p.) injection (note that the therapeutic administration is oral) to groups of TO mice on gestation day (GD) 7 or 8. Fetuses were collected on GD 18. Maternal toxic effects including a dose‐related mortality, a high incidence of abortion, embryo lethality, congenital malformations and intrauterine growth retardation (IUGR) were observed in the LTG‐treated group. Administration of LTG in multiple low doses resulted in a better maternal survival and increased incidence of embryonic resorption and malformations with increasing dose; IUGR was significant but not dose‐dependent. The malformations characteristic of the LTG multiple low dose group fetuses included maxillary‐mandibular hypoplasia, exencephaly, cleft palate, median facial cleft, urogenital anomalies and varying degrees of caudal regression. Skeletal malformations and developmental delay of the skeleton were observed both in single and multiple dose groups. CONCLUSIONS: The results of this study indicate that LTG administered i.p. at high doses can induce intrauterine growth retardation and at low multiple doses causes a dose‐dependent increase in embryonic resorption, craniofacial and caudal malformations as well as maternal toxicity in the mouse. Previous studies in other laboratories have used oral route of exposure and concluded that there are no teratogenic effects of LTG at dose levels that are not maternally toxic. Birth Defects Res B 68:428–438, 2003. © 2003 Wiley‐Liss, Inc.     

A profibrotic function of IL-12p40 in experimental pulmonary fibrosis

The p40 subunit of IL-12 (IL-12p40), but not the heterodimeric form IL-12p70, is secreted during the development of silica-induced lung fibrosis in C57BL/6 mice. To delineate the contribution of IL-12p40 to the lung inflammatory and fibrotic processes, we compared the pulmonary responses with silica particles of IL-12p35-deficient mice (IL-12p35(-/-), able to produce IL-12p40) and IL-12p40-deficient mice (IL-12p40(-/-)). IL-12p35(-/-) and IL-12p40(-/-) animals developed strikingly contrasting responses to silica in comparison with wild-type C57BL/6 mice. Although the IL-12p40(-/-) mice exhibited limited inflammatory and fibrotic reactions, the IL-12p35(-/-) mice presented a robust and well-developed pulmonary inflammation and fibrosis. Furthermore, the silica-induced increase in lung IL-12p40 content was significantly higher in IL-12p35(-/-) mice than in wild-type controls, and was associated with extensive lung fibrosis and pulmonary macrophage infiltration. The contrasting responses observed between these two IL-12 subunit-deficient murine strains were not accompanied by a strict type 1 or type 2 polarization as estimated by the measurements of lung IFN-gamma/IgG2a and IL-4/IgG1 content. In vitro proliferation, type I collagen expression, as well as myofibroblast differentiation of purified pulmonary fibroblasts were not affected by treatment with exogenous rIL-12p40. In vivo, supplementation with rIL-12p40 restored the impaired pulmonary fibrotic response and macrophage accumulation in silica-treated IL-12p40(-/-) mice, and also promoted fibrosis and macrophage influx in wild-type mice. Together, our data suggest that IL-12p40 plays an important role in silica-induced pulmonary inflammation and fibrosis, possibly by exacerbating macrophage recruitment.     

Developmental toxicity assessment of d,l-methylphenidate and d-methylphenidate in rats and rabbits

BACKGROUND: Previous investigations reported no teratogenicity for methylphenidate (MPH). These studies investigated potential teratogenicity of d‐MPH and d,l‐MPH as commitments to the FDA. METHODS: Rabbits received 15, 50, 150 mg/kg/day (mkd) d‐MPH or 20, 60, 200, 300 mkd d,l‐MPH on gestation days 7–20. Rats received 2.5, 10, 40 mkd d‐MPH, or 7, 25, 75, 80 mkd d,l‐MPH on gestation days 6–17. RESULTS: d‐MPH—In rabbits, mortality occurred at 150 mkd. Dilated pupils, increased activity, biting/chewing, respiration, and salivation occurred at ≥15 mkd in rabbits and ≥10 mkd in rats. Decreased food consumption occurred at 40 mkd in rats. Decreased body weight parameters occurred at 150 mkd in rabbits and ≥10 mkd in rats. There were no fetal findings in rabbits. In rats, skeletal variations occurred at 40 mkd. d,l‐MPH—In rabbits, mortality occurred at ≥200 mkd. Dilated pupils, increased activity, biting/chewing, respiration, and salivation occurred at ≥20 mkd in rabbits and ≥25 mkd in rats. Decreased food consumption occurred at ≥200 mkd in rabbits and ≥25 mkd in rats. Decreased body weight parameters occurred at ≥200 mkd in rabbits and ≥25 mkd in rats. In rabbits, two fetuses (separate litters) had spina bifida and malrotated hindlimbs at 200 mkd. In rats, skeletal variations occurred at ≥75 mkd. CONCLUSIONS: There was no teratogenicity with d‐MPH. There was a low teratogenic risk with d,l‐MPH in only the rabbit. Higher C_max may explain differences in results from previous studies. Birth Defects Res (Part B) 83:489‐501, 2008. © 2008 Wiley‐Liss, Inc.