Evaluation of an in vitro three-dimensional HepaRG spheroid model for genotoxicity testing using the high-throughput CometChip platform.

Three-dimensional (3D) culture systems are increasingly being used for genotoxicity studies due to improved cell-to-cell interactions and tissue-like structures that are limited or lacking in 2D cultures. The present study optimized a 3D culture system using metabolically competent HepaRG cells for in vitro genotoxicity testing. 3D HepaRG spheroids, formed in 96- or 384-well ultra-low attachment plates, were exposed to various concentrations of 34 test articles, including 8 direct-acting and 11 indirect-acting genotoxicants/carcinogens as well as 15 compounds that show different genotoxic responses in vitro and in vivo. DNA damage was evaluated using the high-throughput CometChip assay with con-current cytotoxicity assessment by the ATP assay in both 2D and 3D cultures. 3D HepaRG spheroids maintained a stable phenotype for up to 30 days with higher levels of albumin secretion, cytochrome P450 gene expression, and enzyme activities compared to 2D cultures. 3D spheroids also demonstrated a higher sensitivity than 2D cultures for detecting both direct- and indirect-acting genotoxicants/carcinogens, indicating a better prediction of in vivo genotoxicity responses. When DNA damage dose-response data were quantified using PROAST software, 3D spheroids generally had lower or similar benchmark dose values compared to 2D HepaRG cells and were more comparable with primary human hepatocytes. These results demonstrate that 3D models can be adapted to the CometChip technology for high-throughput genotoxicity testing and that 3D HepaRG spheroids may be used as a reliable and pragmatic in vitro approach to better support the hazard identification and risk assessment of potential human genotoxic carcinogens.

Genotoxicity evaluation using primary hepatocytes isolated from rhesus macaque (Macaca mulatta).

Non-human primates (NHPs) have played a vital role in fundamental, pre-clinical, and translational studies because of their high physiological and genetic similarity to humans. Here, we report a method to isolate primary hepatocytes from the livers of rhesus macaques (Macaca mulatta) after in situ whole liver perfusion. Isolated primary macaque hepatocytes (PMHs) were treated with various compounds known to have different pathways of genotoxicity/carcinogenicity and the resulting DNA damage was evaluated using the high-throughput CometChip assay. The comet data were quantified using benchmark dose (BMD) modeling and the BMD50 values for treatments of PMHs were compared with those generated from primary human hepatocytes (PHHs) in our previous study (Seo et al. Arch Toxicol 2020, 2207-2224). The results showed that despite varying CYP450 enzyme activities, PMHs had the same sensitivity and specificity as PHHs in detecting four indirect-acting (i.e., requiring metabolic activation) and seven direct-acting genotoxicants/carcinogens, as well as five non-carcinogens that are negative or equivocal for genotoxicity in vivo. The BMD50 estimates and their confidence intervals revealed species differences for DNA damage potency, especially for direct-acting compounds. The present study provides a practical method for maximizing the use of animal tissues by isolating primary hepatocytes from NHPs. Our data support the use of PMHs as a reliable surrogate of PHHs for evaluating the genotoxic hazards of chemical substances for humans.

Observations of mortality in farmed bison in the Canadian prairies: 2103 - 2016.

The present study is a continuation of a previous mortality study on Saskatchewan bison farms with special emphasis on Malignant Catarrhal Fever. The updated objective of the study was to estimate the most common causes of mortality in farmed bison herds in Western Canada. Results were compared to the previous Saskatchewan study to assess the similarities and differences in the etiology associated with farmed bison deaths across the Prairie Provinces of Canada. The most common cause of death was respiratory disease associated with Mycoplasma bovis, although this was restricted to Alberta and Saskatchewan farm locations. This was in contrast to the previous Saskatchewan based study which did not identify any deaths involving this pathogen. An updated overall assessment of the risks of Malignant Catarrhal Fever in farmed bison at various proximities to sheep operations further confirmed the low risk of occurrence on farms within a 1 km boundary fence distance.

Septicemic pasteurellosis in farmed elk (Cervus canadensis) in Alberta.

Septicemic pasteurellosis is a bacterial disease of domestic and wild animals including bison, elk, and pronghorn antelope caused by Pasteurella multocida. Here we report 2 cases of septicemic pasteurellosis in farmed elk. Pasteurella multocida serogroup B was isolated from multiple tissues in both animals. Gene sequencing (16S ribosomal RNA) and BLAST query confirmed that the sequence is 99% to 100% homologous to the P. multocida sequences in the database.

Pasteurellose septicémique chez des wapitis d'élevage(Cervus canadensis)en Alberta. La pasteurellose septicémique est une maladie bactérienne des animaux domestiques et sauvages, dont le bison, le wapiti et l'antilocarpe, qui est causée par Pasteurella multocida. Dans le présent article, nous présentons un rapport sur 2 cas de pasteurellose septicémique chez les wapitis d'élevage. Le sérogroupe B de Pasteurella multocida a été isolé dans des plusieurs tissus des deux animaux. Le séquençage des gènes (ARN ribosomique16S) et une recherche BLAST a confirmé que la séquence est de 99 % à 100 % homologue aux séquences de P. multocida dans la base de données.(Traduit par Isabelle Vallières).

Demonstration of synchrotron x-ray phase contrast imaging computed tomography of infiltrative transitional cell carcinoma of the prostatic urethra in a dog.

Prostatic urethral transitional cell carcinoma with prostatic invasion in a dog was imaged with abdominal radiography and abdominal ultrasonography antemortem. Synchrotron in-line x-ray phase contrast imaging computed tomography (XPCI-CT) was performed on the prostate ex vivo at the Canadian Light Source Synchrotron and compared to histology. XPCI-CT imaging provides greater soft tissue contrast than conventional absorption-based x-ray imaging modalities, permitting visualization of regions of inflammatory cell infiltration, differentiation of invasive versus noninvasive tumor regions, and areas of necrosis and mineralization. This represents the first report of XPCI-CT images of an invasive prostatic urothelial neoplasm in a dog.

Thyroid hormone concentrations in systemic circulation and ovarian follicular fluid of cows.

The aim of this study was to determine and compare the concentrations of total (T) and free (F) fractions of thyroid hormones (T(3)-triiodithyronine and T(4)-thyroxin) in peripheral circulation and follicular fluid of cows in relation to ovarian follicular status in vivo (Experiment 1), and in the follicles from the slaughterhouse ovaries (Experiment 2). In Experiment 1, estrus was synchronized in 15 cows using two Estrumate (cloprostenol sodium) injections (250 mg cloprostenol intramuscular), the time of ovulation (Day 0) was confirmed by ultrasonography, and ovarian antral follicles were ablated on Day 5. The ensuing superovulatory treatment consisted of eight Folltropin-V injections (50 mg intramuscular) administered twice daily from Day 6 to Day 9, followed by two injections of Estrumate (Day 10 am and pm) and a single dose of Lutropin Alfa (Day 11; 750 IU intramuscular). On Day 5, both TT(3) and FT(3) concentrations were greater (P < 0.05) in serum than follicular fluid from dominant (DFs) or subordinate antral follicles (SFs), and TT(4) concentrations were greater (P < 0.05) in DFs compared with SFs. Serum concentrations of FT(4) were greater (P < 0.05) on Day 12 than on Day 5, and TT(4) concentrations in follicular fluid collected on Day 12 were higher than those in DFs and SFs on Day 5. In Experiment 2, there were no differences (P > 0.05) in thyroid hormone concentrations between the largest and all remaining antral follicles visible on the surface of the ovary (n = 20 ovaries). We concluded that: (i) physiological status of bovine antral follicles (i.e. dominant versus subordinate) may impinge on the accumulation of TT(4) in follicular fluid; and (ii) hormonal ovarian superstimulation increases circulating levels of FT(4) and follicular fluid content of TT(4).

Superovulatory response in a bovine model of reproductive aging.

Two experiments were done to test the hypotheses that aging in cattle is associated with a reduced number of follicles recruited into an ovarian follicular wave, and a reduction in the ovarian response to gonadotropin treatment. Older cows (13-16 years of age) and their daughters (3-6 years of age) were treated with FSH for ovarian superstimulation four times over two consecutive years (31 and 33 superstimulations in old and young cows, respectively, experiments and years combined). In Experiment 1, ovulation was induced using LH. In Experiment 2, cumulus-oocyte complexes were collected by ultrasonographic-guided follicle aspirations before expected ovulations. The ovarian follicular and ovulatory responses were monitored daily by ultrasonography. Fewer 2-5mm follicles (P<0.01) were detected at the expected time of follicular wave emergence in older cows than in their daughters. After superstimulation, older cows had fewer follicles >or=6mm (P<0.01), and tended (P=0.1) to have fewer ovulations than their daughters (32+/-4 versus 40+/-3, respectively). There was a positive correlation in the response of individual cows to successive superstimulatory treatments (r>0.8; P<0.0001) and the number of detected ovulations from one year to the next (r=0.6; P=0.04). In conclusion, aging was associated with fewer 2-5mm follicles at follicular wave emergence and a lesser follicular and ovulatory response after superstimulatory treatment. The follicular and ovulatory response after superstimulation was repeatable within individuals, regardless of age.

Oocyte developmental competence in a bovine model of reproductive aging.

The study was designed to test the hypothesis that aging in cattle is associated with reduced developmental competence of oocytes. The hypothesis was tested by comparing embryo production and pregnancy rates between 13- to 16-year-old cows (n = 6 in Year 1 and n = 9 in Year 2) and their 3- to 6-year-old young daughters (n = 8 in Year 1 and n = 9 in Year 2) after superovulation and transfer of embryos into an unrelated group of young recipients. Embryos were transferred into 2- to 5-year-old recipient cows (n = 99) as singletons (n = 45) or in pairs (n = 54 pairs). Embryo survival in recipients was determined by ultrasonography and by the number of calves born. Between old versus young cows, the number of ovulations (31 +/- 4 vs 38 +/- 3; P = 0.2) and the number of corpora lutea (25 +/- 3 vs 29 +/- 2; P = 0.3) did not differ, but fewer (P = 0.04) embryos were recovered from old cows (6 +/- 2) than their daughters (12 +/- 2). A higher proportion (P < 0.0001) of unfertilized oocytes/uncleaved zygotes were recovered from old cows (222/312, 71%) than their daughters (119/316, 38%). Among the embryos recovered, the proportion of International Embryo Transfer Society Grades 1-2 embryos was similar (P = 0.9) between old (59/90, 66%) and young cows (130/194, 67%). The survival of embryos after transfer into recipients, and the proportion of calves born were also similar between old and young cows. In conclusion, recovery of fewer embryos and a greater proportion of unfertilized oocytes/uncleaved zygotes suggest reduced developmental competence of oocytes from old cows, but there was no difference between age groups in embryo survival after the morula/blastocyst stage.

Bovine model of reproductive aging: response to ovarian synchronization and superstimulation.

The responsiveness of the hypothalamo-pituitary axis to steroid treatments for ovarian synchronization and the ovarian superstimulatory response to exogenous FSH was compared in 13-14 year old cows and their 1-4 year old young daughters. We tested the hypotheses that aging in cattle is associated with: (1) decreased follicular wave synchrony after estradiol and progesterone treatment; (2) delayed LH surge and ovulation in response to exogenous preovulatory estradiol treatment; (3) reduced superstimulatory response to exogenous FSH. Higher plasma FSH concentrations (P<0.01), and a tendency (P=0.07) for fewer 4-5 mm follicles at wave emergence were observed in old cows (n=10) than in young cows (n=9). The suppressive effect of estradiol/progesterone treatment on FSH was similar between old and young cows. Although the preovulatory LH surge in response to estradiol treatment was delayed in old than young cows (P=0.01), detected ovulation times were not different. No difference in ovarian superstimulatory response was detected between age groups, but old cows (n=8) tended (P=0.10) to have fewer large follicles (>or=9 mm) 12 h after last FSH treatment than in young cows (n=7). We concluded that pituitary and ovarian responsiveness to estradiol/progesterone synchronization treatment was similar between old and young cows, but aging was associated with a delayed preovulatory LH surge subsequent to estradiol treatment. Old cows tended to have fewer large follicles after superstimulatory treatment than young cows.

Bovine model for the study of reproductive aging in women: follicular, luteal, and endocrine characteristics.

At present, there is no well-characterized animal model to study the effects of aging on fertility in women. The objectives of the study were to characterize age-related changes in ovarian and endocrine functions in old cows and to investigate the validity of a bovine model for the study of human reproductive aging. We tested the hypotheses that aging in cattle is associated with 1) elevated concentrations of gonadotropins and reduced concentrations of steroid hormones in systemic circulation and 2) increased recruitment of ovarian follicles during wave emergence. Daily ultrasonography was performed in 13- to 14-yr-old cows (n = 10) and their 1- to 4-yr-old daughters (n = 9) for one interovulatory interval to study ovarian function. Plasma samples were obtained every 12 h for determination of FSH, LH, progesterone, and estradiol concentrations. Circulating FSH concentrations were higher (P = 0.009) during follicular waves in old cows than in their daughters, but the number of 4- to 5-mm follicles recruited into a wave was lower (P = 0.04) in old cows. Plasma LH concentrations did not differ between groups (P = 0.4), but the ovulatory follicle in two-wave cycles was smaller in old cows (P = 0.04). Plasma estradiol concentrations were higher (P = 0.01) in old cows, and luteal phase progesterone tended to be lower (P = 0.1). We conclude that these changes are consistent with those reported for women approaching menopause transition. Therefore, our results validate the use of the bovine model to study reproductive aging in women.