Surrogate Humane Endpoints in Small Animal Models of Acute Lung Injury: A Modified Delphi Consensus Study of Researchers and Laboratory Animal Veterinarians.

OBJECTIVES: In many jurisdictions, ethical concerns require surrogate humane endpoints to replace death in small animal models of acute lung injury. Heterogenous selection and reporting of surrogate endpoints render interpretation and generalizability of findings between studies difficult. We aimed to establish expert-guided consensus among preclinical scientists and laboratory animal veterinarians on selection and reporting of surrogate endpoints, monitoring of these models, and the use of analgesia. DESIGN: A three-round consensus process, using modified Delphi methodology, with researchers who use small animal models of acute lung injury and laboratory animal veterinarians who provide care for these animals. Statements on the selection and reporting of surrogate endpoints, monitoring, and analgesia were generated through a systematic search of MEDLINE and Embase. Participants were asked to suggest any additional potential statements for evaluation. SETTING: A web-based survey of participants representing the two stakeholder groups (researchers, laboratory animal veterinarians). Statements were rated on level of evidence and strength of support by participants. A final face-to-face meeting was then held to discuss results. SUBJECTS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Forty-two statements were evaluated, and 29 were rated as important, with varying strength of evidence. The majority of evidence was based on rodent models of acute lung injury. Endpoints with strong support and evidence included temperature changes and body weight loss. Behavioral signs and respiratory distress also received support but were associated with lower levels of evidence. Participants strongly agreed that analgesia affects outcomes in these models and that none may be necessary following nonsurgical induction of acute lung injury. Finally, participants strongly supported transparent reporting of surrogate endpoints. A prototype composite score was also developed based on participant feedback. CONCLUSIONS: We provide a preliminary framework that researchers and animal welfare committees may adapt for their needs. We have identified knowledge gaps that future research should address.

Source of a micro-nutrient in a semi-synthetic basal diet as a causative factor in inducing urinary calculi in rats and its inhibition by PSC 833, a potent inhibitor of P-glycoprotein.

We report a serendipitous finding of urinary calculi in rats fed a semi-synthetic basal diet. This observation was made during ongoing studies to evaluate the inhibitory effect of PSC 833, a potent inhibitor of P-glycoprotein, on development of tumors in rodent tumor model systems. A large number of specific-pathogen-free (SPF) female Sprague-Dawley and SPF male Fischer 344 rats being fed the diet were euthanized when it became evident clinically that they were uremic. At necropsy, the renal pelvis, ureters, and urinary bladder contained numerous calculi. The presence of urinary calculi was determined to be related to the source of a Food Chemical Codex grade of choline bitartrate. Rats being fed the same basal diet containing the United States Pharmacopia grade of choline bitartrate failed to develop urinary calculi. Interestingly, rats treated with the P-glycoprotein inhibitor were at significantly reduced risk of developing urinary calculi. This finding highlights how something seemingly innocuous as a minor dietary constituent can have a profound impact and, thereby, affect experimental outcome.

Folic acid supplementation promotes mammary tumor progression in a rat model.

Folic acid supplementation may prevent the development of cancer in normal tissues but may promote the progression of established (pre)neoplastic lesions. However, whether or not folic acid supplementation can promote the progression of established (pre)neoplastic mammary lesions is unknown. This is a critically important issue because breast cancer patients and survivors in North America are likely exposed to high levels of folic acid owing to folic acid fortification and widespread supplemental use after cancer diagnosis. We investigated whether folic acid supplementation can promote the progression of established mammary tumors. Female Sprague-Dawley rats were placed on a control diet and mammary tumors were initiated with 7,12-dimethylbenza[a]anthracene at puberty. When the sentinel tumor reached a predefined size, rats were randomized to receive a diet containing the control, 2.5x, 4x, or 5x supplemental levels of folic acid for up to 12 weeks. The sentinel mammary tumor growth was monitored weekly. At necropsy, the sentinel and all other mammary tumors were analyzed histologically. The effect of folic acid supplementation on the expression of proteins involved in proliferation, apoptosis, and mammary tumorigenesis was determined in representative sentinel adenocarcinomas. Although no clear dose-response relationship was observed, folic acid supplementation significantly promoted the progression of the sentinel mammary tumors and was associated with significantly higher sentinel mammary tumor weight and volume compared with the control diet. Furthermore, folic acid supplementation was associated with significantly higher weight and volume of all mammary tumors. The most significant and consistent mammary tumor-promoting effect was observed with the 2.5x supplemental level of folic acid. Folic acid supplementation was also associated with an increased expression of BAX, PARP, and HER2. Our data suggest that folic acid supplementation may promote the progression of established mammary tumors. The potential tumor-promoting effect of folic acid supplementation in breast cancer patients and survivors needs further clarification.

Effect of rosiglitazone on bone quality in a rat model of insulin resistance and osteoporosis.

OBJECTIVE: Rosiglitazone (RSG) is an insulin-sensitizing drug used to treat type 2 diabetes mellitus. The A Diabetes Outcome Progression Trial (ADOPT) shows that women taking RSG experienced more fractures than patients taking other type 2 diabetes drugs. These were not osteoporotic vertebral fractures but, rather, occurred in the limbs. The purpose of this study was to investigate how RSG treatment alters bone quality, which leads to fracture risk, using the Zucker fatty rat as a model. RESEARCH DESIGN AND METHODS: A total of 61 female 4-month-old rats were divided into six groups. One Sham group was a control and another was administered oral RSG 10 mg/kg/day. Four ovariectomized (OVX) groups were dosed as follows: controls, RSG 10 mg/kg, alendronate (ALN, injected at 0.7 mg/kg/week), and RSG 10 mg/kg plus ALN. After 12 weeks of treatment, bone quality was evaluated by mechanical testing. Microarchitecture, bone mineral density (BMD), cortical bone porosity, and bone remodeling were also measured. RESULTS: OVX RSG 10 mg/kg rats had lower vertebral BMD and compromised trabecular architecture versus OVX controls. Increased cortical bone porosity and decreased mechanical properties occurred in these rats. ALN treatment prevented decreased BMD and architectural and mechanical properties in the OVX model. Reduced bone formation, increased marrow adiposity, and excess bone resorption were observed in RSG-treated rats. CONCLUSIONS: RSG decreases bone quality. An unusual finding was an increase in cortical bone porosity induced by RSG, consistent with its effect on long bones of women. ALN, an inhibitor of bone resorption, enhanced mechanical strength and may provide an approach to partially counter the deleterious skeletal effects of RSG.

Effect of maternal and postweaning folic acid supplementation on mammary tumor risk in the offspring.

Intrauterine and early life exposure to folic acid has significantly increased in North America owing to folic acid fortification, widespread supplemental use, and periconceptional supplementation. We investigated the effects of maternal and postweaning folic acid supplementation on mammary tumor risk in the offspring. Female rats were placed on a control or folic acid-supplemented diet prior to mating and during pregnancy and lactation. At weaning, female pups from each maternal diet group were randomized to the control or supplemented diet and mammary tumors were induced with 7,12 dimethylbenz[a]anthracene at puberty. At necropsy, mammary tumor parameters, genomic DNA methylation, and DNA methyltransferase activity were determined in the offspring. Both maternal and postweaning folic acid supplementation significantly increased the risk of mammary adenocarcinomas in the offspring (OR = 2.1, 95% CI 1.2-3.8, P = 0.008 and OR = 1.9, 95% CI 1.1-3.3, P = 0.03, respectively). Maternal folic acid supplementation also significantly accelerated the rate of mammary adenocarcinoma appearance (P = 0.002) and increased the multiplicity of mammary adenocarcinomas (P = 0.008) in the offspring. Maternal, but not postweaning, folic acid supplementation significantly reduced global DNA methylation (P = 0.03), whereas postweaning, but not maternal, folic acid supplementation significantly decreased DNA methyltransferase activity (P = 0.05) in nonneoplastic mammary glands of the offspring. Our findings suggest that a high intrauterine and postweaning dietary exposure to folic acid may increase the risk of mammary tumors in the offspring. Further, they suggest that this tumor-promoting effect may be mediated in part by altered DNA methylation and DNMT activity.

Effects of dietary folate on the development and progression of mammary tumors in rats.

Epidemiologic studies have suggested that dietary intake and blood levels of folate may be inversely related to the risk of breast cancer. However, epidemiologic evidence has not been consistent nor has it provided unequivocal support for this purported inverse relationship. Recent evidence has also raised a concern that folate supplementation may promote carcinogenesis if provided after neoplastic foci are established in the target organ. This study investigated the effect of dietary folate deficiency and supplementation on the development and progression of mammary tumors in the N-methyl-N-nitrosourea (MNU) rat model. Weanling, female Sprague-Dawley rats were fed diets containing 0, 2 (control) or 8 mg folic acid/kg diet during the initiation or the promotion phase of MNU-induced mammary tumorigenesis. At necropsy, all macroscopic mammary tumors were identified and histologically confirmed. Dietary folate deficiency and supplementation provided during the initiation phase did not significantly modulate the development of mammary tumors. In contrast, dietary folate deficiency provided during the promotion phase significantly inhibited the rate of appearance, incidence, mean volume and weight of adenocarcinomas compared with the control and supplemental diets. Folate supplementation provided during the promotion phase did not significantly modulate mammary tumorigenesis compared with the control group. These data indicate that moderate folate deficiency inhibits, whereas dietary folate supplementation at four times the basal dietary requirement does not promote, the progression of MNU-induced mammary neoplastic foci in this rat model. However, the limitations associated with the route and dose of MNU administration preclude a definitive conclusion concerning the effect of folate status on the initiation of MNU-induced mammary tumorigenesis.

Dietary folate deficiency suppresses N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats.

Epidemiologic studies have suggested that dietary folate intake is inversely related to breast cancer risk. However, epidemiologic evidence has not been consistent nor has it provided unequivocal support for this purported inverse relationship. This study investigated the effect of dietary folate on N-methyl-N-nitrosourea (MNU)-induced mammary tumorigenesis in rats. Weanling, female Sprague-Dawley rats were fed diets containing either 0 (deficient; n = 22), 2 (basal dietary requirement, control; n = 20) or 8 mg (supplemented; n = 20) folate/kg diet for 30 weeks. At 50 days of age, rats received an i.p. injection of MNU (50 mg/kg body wt). At necropsy, all macroscopic mammary tumors were identified and examined microscopically. The effect of dietary folate on genomic DNA methylation in mammary tumorigenesis was determined by the in vitro methyl acceptance assay. The incidence of mammary adenoma and adenocarcinoma in the folate-deficient group was lower than that of the control and folate-supplemented groups (55 versus 90 and 75%, respectively, P = 0.043). Kaplan-Meier analyses also demonstrated a similar trend in the rates of appearance of either adenoma or adenocarcinoma (P = 0.06). In contrast, folate supplementation did not significantly modulate mammary tumorigenesis compared with the control group. Although mammary tumors were significantly hypomethylated compared with non-neoplastic mammary tissues in each dietary group (P < 0.03), folate status did not significantly affect the extent of DNA methylation. The data suggest that dietary folate deficiency of a moderate degree suppresses, whereas folate supplementation at four times the basal dietary requirement does not significantly modulate, mammary tumorigenesis in this model. The role of folate in mammary tumorigenesis needs to be clarified for safe and effective prevention of breast cancer.