Effects of food restriction on toxicological parameters in juvenile rats

Abstract

To examine the effects of decreased food consumption on toxicological parameters in juvenile rats, rats on postnatal day 21 were fed 40%, 50% (only four weeks), and 60% less food, compared to that of controls for four or eight weeks, and clinical observations, measurement of body and organ weights, morphological differentiation analysis, clinical pathology, and macroscopic and microscopic examinations were conducted. The body weight decreased depending on the degree of food restriction (FR). Cleavage of the balano-preputial skinfold was delayed, and cell debris in the epididymal lumen was noted as a related finding after four-week FR. Vaginal opening was also delayed, and some histopathological findings, such as absence of corpus luteum in the ovary, mucinous degeneration in the vagina, and immature uterus, were noted after eight-week FR. Erythrocyte count increased after four-week FR, but slightly decreased in males only after eight-week FR, and decreased leukocyte and/or reticulocyte counts, accompanied by related histopathological findings were noted after four- and eight-week FR. In blood chemistry, the levels of total protein including globulin, glucose, triglyceride, and calcium decreased, and sodium and chloride increased after four- and eight-week FR. Increases in activities of aspartate transaminase and lactate dehydrogenase and total bilirubin levels were noted after four-week FR, which were attenuated after eight-week FR. The effects of FR seemed to be more remarkable after four weeks. In drug safety evaluation, findings caused by malnutrition should be considered in juvenile toxicity studies when decreased food consumption is observed.

INTRODUCTION

Pediatrics is different from adult care as children and juveniles have not completed the process of development and growth into adults, and several organs are functionally and/or anatomically immature (Picut and Remik, 2019). Therefore, in nonclinical drug development, when test compounds are administered, differential effects may be induced in juvenile and adult animals. As International Conference on Harmonization (ICH) S11 has recently been issued, juvenile animal studies are important to support pediatric drug development. Clinical observation, growth, food consumption, sexual development, clinical pathology, and anatomical pathology have been listed as core endpoints of this type of study (2020).

Body weight and food consumption are the major endpoints in toxicological evaluation. Decreases in these parameters occur frequently as toxicological changes that can affect other endpoints such as clinical and anatomical pathology. The effects of FR have been examined in animals used for general toxicity studies. In these reports (Moriyama et al., 2008; Levin et al., 1993; Oishi et al., 1979), the age of animals ranged between 5–8 weeks, and feeding was restricted up to 75% for 2–4 weeks. As a consequence, low body and organ weights (specifically the liver), and/or low nutritional parameters in blood chemistry, including triglyceride, total protein, and inorganic phosphorus, were noted. Furthermore, a slight decrease in hematopoietic tissues, such as leukocyte and bone marrow, was observed, although hemoconcentration was noted.

However, reports regarding the effects of FR on toxicological parameters of juvenile rats are scanty. Therefore, we conducted an FR study using three-week-old rats (following weaning so that feeding could be controlled), an age corresponding to that of a two-year-old human (Picut and Remik, 2019). The effects of malnutrition induced by FR during postnatal growth and development were investigated using general toxicological endpoints, such as clinical signs, body weight, hematology, blood chemistry, organ weights, and macroscopic and microscopic examinations. Other than general toxicological endpoints, sexual maturation (physical developments) was also investigated. The FR period was set for four weeks, and after the restriction period, the animals were seven weeks old, an age when the main organs of an intact rat are morphologically mature (Picut and Remik, 2019). In addition, an eight-week FR was conducted to investigate the chronic effects of malnutrition. Knowledge of parameters related to low body weight and/or food consumption is useful for future evaluation of test compound profiles in juvenile animals to distinguish the effects of compounds or secondary effects of malnutrition.

MATERIALS AND METHODS

Animals

Twenty-eight pregnant Crl:CD(SD) rats [gestation day (GD) 14] were obtained from Charles River Japan, Inc. (Kanagawa, Japan) and delivered naturally. The pups used in this study were determined as follows. The day of delivery was defined as postnatal day (PND) 0, and on PND 1, pups with no external abnormalities were randomly selected from each litter. The total number of pups in each litter was adjusted to five each of males and females. Allocation of pups of the same sex from the same litter was avoided, and dams with no abnormal nursing conditions were selected for cross-fostering.

After weaning, the rats were housed in hanging metal and mesh-bottom cages in animal rooms. Room conditions were controlled as follows: temperature 22 ± 3°C, relative humidity 50 ± 20%, air exchange 15 times/hr, and a 12-hr light/dark cycle (lights on from 7:00 to 19:00). Animals were allowed free access to tap water. This study was approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd. (Approval Nos. IACUC010-310 and IACUC010-322) and was performed in accordance with the animal welfare bylaws of Shin Nippon Biomedical Laboratories, Ltd., Drug Safety Research Laboratories, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Feeding restriction study

Four males and four females were randomly selected from each litter to standardize the number of pups on PND 4 and weaned on PND 21. On PND 21, males and females were allocated to 4 and 3 groups consisting of 10 animals/sex/group for the four- and eight-week FR study, respectively. Animals were fed 0%, 40%, 50%, and 60% less food for the four-week FR study and 0%, 40%, and 60% less food for the eight-week FR study from PND 21. For the FR groups, the feeding weights on PND 21 were calculated based on food consumption in the control group from a previous internal study (data not shown). The feeding weights from PND 22 onward were calculated for each sex every day based on food consumption data from the previous day’s values for the control group. The 4 week of food restriction duration was set following in the general toxicity studies, and the two-fold duration of 8 weeks was also set. Solid food (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) was provided ad libitum to control animals. Overnight fasting prior to necropsy was conducted for the eight-week FR study only. The study design is shown in Fig. 1.

Fig. 1

Study design.

Animals were observed for survival and physical conditions and were weighed each day. In males, cleavage of the balano-preputial skinfold was observed each day from PND 45 until study completion. For females, vaginal opening was observed on PND 35 and each day from PND 40 each day until study completion.

Clinical pathology

At the scheduled necropsy, animals were anesthetized using sodium pentobarbital (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) to obtain blood samples and then euthanized by exsanguination. Blood samples for the hematology were treated with an anticoagulant EDTA-2K. Blood samples for the coagulation tests were treated with a syringe containing 100 μL of 3.8 w/v% sodium citrate and centrifuged (room temperature, 1700 × g, 10 min) to obtain plasma. Blood samples for clinical chemistry analyses were centrifuged (room temperature, 1700 × g, 10 min) to obtain serum. The hematological parameters were as follows: erythrocyte count (RBC), leukocyte count (WBC), hematocrit (HCT), hemoglobin concentration (HGB), platelet count (PLT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), calculated distribution width of erythrocyte coefficient of variation (RDW-CV), reticulocyte count (RET), differential leukocytes, prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (fibrinogen) using the fully automated hematology analyzer (XT-2000iV, Sysmex Corporation) or the automatic blood coagulation analyzer (CS-5100, Beckman Coulter Inc.). The blood chemistry parameters were as follows: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT), creatine kinase (CK), glutamate dehydrogenase (GLDH), total bilirubin (T-Bil), total protein (TP), albumin, globulin, total cholesterol (T-Cho), triglyceride (TG), glucose, urea nitrogen (UN), creatinine (Cre), inorganic phosphorus (IP), calcium (Ca), sodium (Na), potassium (K), and chloride (Cl) using the automatic analyzer (JCA-BM6070, JOEL Ltd.) or the automatic electrophoresis apparatus (AES320, Beckman Coulter Inc.).

Histopathology

For each animal, the external appearance and all organs/tissues in the thoracic, abdominal, and cranial cavities were visually examined, and the lungs, liver, heart, kidneys, testis, epididymides, prostate, seminal vesicles, ovaries, pituitary gland, brain, spleen, thymus, and adrenal glands were weighed, fixed in 10 vol% neutral buffered formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE) and examined microscopically. The trachea, tongue, submandibular glands, esophagus, stomach, intestine including Peyer's patches, pancreas, aorta, urinary bladder, uterus, vagina, spinal cord, sciatic nerves, bone marrow, lymph node, thyroids, lacrimal glands, Harderian glands, skeletal muscle, skin, and mammary gland were fixed in 10 vol% neutral buffered formalin, embedded in paraffin, sectioned, stained with HE, and examined microscopically. The eyeballs, including optic nerve, were fixed in 3% glutaraldehyde and 2.5% formalin. Crown-rump length (CRL) and right femur length were measured after eight-week FR.

Statistical analysis

Data on the morphological development of reproductive organs were analyzed using Fisher’s exact test (Siegel, 1956).

Numerical data were analyzed using Bartlett’s test (Snedecor and Cochran, 1980) for homogeneity of variance for comparison between the control and FR groups. When the variance was homogeneous, Williams’ test (Williams, 1972), assuming a FR-related trend, was performed. With no significant differences in Williams’ test, Dunnett’s test (Dunnett, 1955) was performed to compare differences in the means between the control and FR groups. When the variance was heterogeneous, the Shirley–Williams test (Shirley, 1977; Williams, 1986) that assumes an FR-related trend was performed. With no significant differences in the Shirley-Williams test, Steel’s test (Steel, 1959) was performed to compare differences in the mean rank between the control and FR groups.

Bartlett’s test was conducted at 5% significance level, Williams’ and Shirley-Williams’ tests were conducted at a two-tailed significance level of 5%, and the other tests were conducted at two-tailed significance levels of 5% and 1%. Each test was performed using SAS System for Windows (version 9.3, SAS Institute Inc., Cary, NC, USA).

RESULTS

Food restriction

Mean feed consumption for the male and female controls gradually increased from 8.5 and 7.3 gm/day, respectively, at the beginning of study to 29.1 and 18.0 gm/day, respectively, on PND 49 and 30.3 and 19.9 gm/day, respectively, on PND 75. The mean food consumption shift in the eight-week FR study is shown in Fig. 2.

Fig. 2

Shift in food consumption (eight-week FR study).

The effects of food restriction on physical characteristics

The in-life data for the four-week FR study are not shown in this report because the data were similar to those of the first four weeks in the eight-week FR study. Although the 50% FR was not set in the eight-week FR study, it was not considered appropriate to compare the results of the 50% FR of four-week FR study to those of the eight-week FR study. None of the rats died or exhibited FR-related adverse clinical signs, and no abnormalities were observed in the clinical observations. Body weight gain was suppressed (Fig. 3A), and the body weight ratio of the FR group with control group decreased gradually till PND 40 (40% FR, 65%) and PND 50 (60% FR, 45%), after which these ratios reached plateau (Fig. 3B).

Fig. 3

(A) Body weight (eight-week FR study),  (B) Body weight ratio vs. control (eight-week FR study).

Morphological differentiation (cleavage of the balano-preputial skinfold and vaginal opening) was delayed depending on the percentage of FR (Fig. 4). Cleavage of the balano-preputial skinfold was completed on PND 51 and PND 63 at 40% and 60% FR, respectively (control, PND 48). Vaginal opening was completed on PND 57 and PND 77 at 40% and 60% FR, respectively (control, PND 40).

Fig. 4

Morphological differentiation (eight-week FR study).

The effects of food restriction on clinical pathology

The results of hematology are presented in Table 1. After four-week FR, increases in RBC, MCHC, and RDW-CV were noticed in both sexes in all restricted groups, except for RBC in the 40% FR female group, and MCHC and RDW-CV in the 40% and 50% FR female groups. Decreases in MCV, MCH, and WBC were noticed in both sexes in all FR groups and reticulocyte count in both sexes in the 50% and 60% FR groups.

Table 1. Summary of hematology.

After eight-week FR, decreases in RBC, HGB, HCT, RET, and WBC were noticed in males in all FR groups. In females, decreases in HGB, HCT, MCV, and MCH in the 60% FR group and platelet count and WBC in all FR groups were noticed. Increases in RDW-CV were also noticed in females in the 60% FR group.

In coagulation tests (Table 1), after four-week FR, prolongation of PT for both sexes in all FR groups (except for females in the 40% FR group), shortening of APTT in females in the 50% and 60% FR groups, and a decrease in fibrinogen for both sexes in all FR groups were observed. After eight-week FR, prolongation of PT and a decrease in fibrinogen were observed in females and both sexes in all FR groups, respectively.

In blood chemistry (Table 2), after four-week FR, increases in AST, T-Bil, Na, Cl, IP, and A/G ratio were noticed in males of all FR groups and in LDH and CK in the 50% and 60% FR groups. Decreases in ALT, ALP, TP, TG, glucose, UN, Ca, K, and globulin were noticed in males of all FR groups, in T-cho in the 50% and 60% FR male groups, and in creatinine in the 60% FR group.

Table 2. Summary of blood chemistry.

For females, increases were observed in AST, Na, Cl, and A/G ratio in all FR groups; in LDH, T-Bil, UN in the 50% and 60% FR groups; and in GLDH in the 60% FR group. Decreases in ALP, TP, TG, glucose, Ca, and globulin levels were noticed in all FR groups.

After eight-week FR, increases were noticed in ALP, T-cho, Na, Cl, and A/G ratios in males of all FR groups, and in AST, GGT in the 60% FR group; decreases were noticed in TP, TG, Ca, and globulin in males of all FR groups and in glucose and K in the 60% FR group. In females, increases in ALP, IP, and Cl, and decreases in TP, glucose, Ca, K, GLDH, and globulin were noticed in all FR groups. Additionally, increases in AST, Na, and A/G ratios were noted in the 60% FR group.

The effects of food restriction on the macroscopic pathology

The results of organ weights are presented in Tables 3 and 4. In macroscopic examination, no effects resulting from FR were observed. The absolute organ weights decreased in all FR groups (<40% vs. control); however, the degree of decrease in the brain weights after four- and eight-week FR (>11% vs. control) and in weights of the testis and epididymides after eight-week FR (-14% and -21% vs. control, respectively) was moderate. The trend of increase or decrease in relative organ-to-brain weights was similar to that of the absolute organ weight mentioned above. Although relative organ-to-body weights for many organs were comparable to or higher than the control values after four- and eight-week FR, relative organ-to-body weights of the pituitary, liver, seminal vesicles, and ovaries were lower than the control values after four-week FR, and low organ-to-body weight values in the liver and ovaries were also noticed after eight-week FR. Additionally, we considered the effects of FR on the CRL and femur length. Low CRL and femur length after eight-week FR were noticed in all FR groups (Table 5). High relative femur length compared to CRL was noted in all FR groups, except for females in the 40% FR group. Overall, FR did not affect the macroscopic pathology in the rat.

Table 3. Organ weight in males.

Table 4. Organ weight in females.

Table 5. Summary of crown-rump and femur lengths after eight-week FR.

The effects of food restriction on the microscopic pathology

The results of organs affected by FR are expressed in Table 6. The following findings were observed after four-week FR: atrophy of the hepatocytes, decreased incidence of extramedullary hematopoiesis in the spleen, atrophy of the adipose tissue in the skin (subcutis), and atrophy of the lacrimal gland acinar cells (decreased size and secretary granules of the acinar cells) were noticed in both sexes, and atrophy of the submandibular gland acinar cells (decreased size of acinar cells) was noted in females in all the FR groups; an absence of the corpus luteum in the ovary, mucinous degeneration in the vagina, and decreased zymogen granules in the pancreatic acinar cells were noted in females in the 50% and 60% FR groups; cell debris in the lumen of the epididymides was noted in males in all the FR groups, and atrophy of the mammary glands was noticed in males in the 60% FR group; immature uterus and decreased cellularity of the bone marrow were noticed in females in the 60% FR group; and atrophy of the submandibular gland acinar cells was noted in males in the 50% FR group. Overall, debilitating findings (atrophy and findings suggesting the decreased hematopoiesis) were observed in many organs after four- and/or eight-week FR.

Table 6. Microscopic findings.

DISCUSSION

A suppression in body weight gain and delayed differentiation of external genitalia were noted; however, body weight loss that was noted in adult rats receiving >25% FR (Moriyama et al., 2008) was not observed in juvenile rats. Several organs were affected more than body weight; therefore, they were considered sensitive to FR. Unchanged or increased relative-to-organ weights were considered to be related to the loss of fat, muscles, and water from the whole body (Levin et al., 1993). In previous reports on young adult rats, statistical significance for absolute brain weights was noted only in one study (Levin et al., 1993), in which animals receiving FR (4–5-week-old) were younger than those in other studies (five- or seven-week-old). The growth velocity of the brain is high at the juvenile stage (Watson et al., 2006), and therefore decreased absolute brain weight might be noted in case of FR from a younger age; however, brain weight is generally considered to be less affected by the changes in other organs. Low CRL and femur length after eight-week FR but high relative femur length compared to body weight and CRL were noted. Therefore, body weight was a more sensitive parameter than CRL or femur length.

In addition to the delay of morphological differentiation, cell debris in epididymides and several findings of female reproductive organs, such as the absence of corpora lutea, uterine immaturity, and degeneration of the vaginal epithelial mucinous, were observed after four- and/or eight-week FR. Maternal food restriction during the perinatal period (Léonhardt et al., 2003) and four-week nutritional dwarfing after weaning (Compagnucci et al., 2002) affect the pituitary–gonadal axis, resulting in delayed puberty in rats. In adult male rats, 30% FR has induced decreases in testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (Abou-Heif et al., 2010), and 50% FR in adult females has resulted in a decreased gene expression of kiss peptin in hypothalamus, LH and FSH in pituitary, and decreased serum concentrations of estrogen, LH, and FSH (Ahmed et al., 2012). Therefore, not only growth retardation but an imbalance in hormone levels was induced by FR, resulting in a delayed morphological differentiation and an occurrence of histopathological findings in female reproductive organs. Cell debris instead of sperm in the epididymidies observed after fourweek-FR was considered to be related to growth retardation and delayed differentiation of the external genitalia. However, a decreased relative body weight, decreased secretion in accessory male genital organs, or degeneration of the seminiferous tubules, which were observed at 25 and/or 50% feeding for two weeks in young adult rats (Moriyama et al., 2008), were not noticed for up to 60% FR in juvenile rats in this study.

Low liver weight, parameters of malnutrition in blood chemistry (decreased TG, TP, and glucose and increased IP), and atrophy of hepatocytes observed in juvenile rats were also noted in young adult rats (aged 5–8 weeks at the time of FR) (Moriyama et al., 2008; Levin et al., 1993; Oishi et al., 1979). Atrophy of hepatocytes observed after four-week FR disappeared after eight-week FR. In the submandibular glands, atrophy of the acinar cells was noticed after four-week FR, and decreased granules in the granular duct were noted after eight-week FR. Histopathological findings in liver (cytoplasmic atrophy and hepatocellular single cell necrosis) and salivary glands (decreased secretion and single cell necrosis) were reported in severe conditions only (25% FR) in young adult rats (Moriyama et al., 2008); therefore, juvenile rats may be more sensitive than older rats. Decreased urea nitrogen in males was noted after four-week FR and was considered to be owing to a larger amino acid requirement at a younger age (White et al., 2000), although the reason for increased urea nitrogen in females was unclear.

A decrease and an increase in ALP levels were noted after four- and eight-week FR, respectively. Plasma ALP is known to decrease after overnight fasting (Martins et al., 2001), and in the present study, the feeding condition before blood sampling was different between four- and eight-week FR, ad libitum vs. fasting. In addition, plasma ALP levels in aged rats are lower than those in younger rats (Wolford et al., 1987). Therefore, the baseline plasma ALP levels were different between the four- and eight-week FR groups. After four-week FR, the FR groups were considered to be in the similar condition of overnight fasting, and to be resulted in a decrease in ALP level. In contrast, eight-week FR resulted in an increase in ALP level, which might be related to growth retardation, causing young animal-like conditions in the FR groups compared to that in the control group.

A high RBC content, but not HGB or HCT, was noticed after four-week FR. A similar phenomenon has been reported in several studies in young adult rats with up to two- or four-week FR (Moriyama et al., 2008;Levin et al., 1993; Miyata et al., 2009), and this phenomenon has been considered to be induced by hemoconcentration and long lifespan of erythrocytes. After eight-week FR, slight decreases in erythrocyte parameters were observed. These inverse changes were considered to be owing to a decreased erythropoietin level induced by FR [observed in young adult rats also (Asanuma et al., 2011)] because decreased incidence of extramedullary hematopoiesis in the spleen and/or decreased cellularity of the bone marrow accompanied by a decrease in RET were observed in both the four- and eight-week FR groups.

Low WBC was noticed after four- and eight-week FR in this study, which was also observed in young adult rats with up to two- or four-week FR (Moriyama et al., 2008; Levin et al., 1993; Miyata et al., 2009). In these studies, decreased cellularity in the bone marrow was noted, and the incidence and severity of this change were related to the level of food restriction (Moriyama et al., 2008; Levin et al., 1993). The degree of change after eight-week FR was milder than that after four-week FR. However, the ratio of each leukocyte compared to that of each control was unchanged after four- or eight-week FR (data not shown), and no histopathological changes in the thymus were noted in any group; therefore, the effects of FR on lymphocytes were considered to be limited.

Additionally, platelet levels decreased after eight-week FR in this study, which was also observed in young adult rats (Moriyama et al., 2008; Levin et al., 1993). Coagulation parameters, such as PT, APTT, and fibrinogen, were affected by FR in this study; however, the prolongation of PT or shortening of APTT were very small, and no related findings, such as bleeding or blood clots, were noticed in clinical, macroscopic, and microscopic observations. Therefore, no significant impact of FR was noticed on the coagulation system.

Suppressed body weight gain was noted and the body weight ratios of the FR group with control group reached plateau. Blood chemistry parameters after four-week FR were considered to indicate the decreased nutritional intake (i.e., TP, Glob, TG, T-cho, glucose, and Ca) and increased energy metabolism (CK, AST, and LDH) to compensate for decreased nutrition. Some effects on the digestive system after four-week FR, including organ weight and histopathological changes in the liver, seemed to be attenuated after eight-week FR. Attenuation in the digestive system is considered to be related to a decreased basal metabolic rate to minimize the negative energy balance during malnutrition (Emery, 2005). Furthermore, some other changes (i.e., LDH, UN) after four-week FR were attenuated after eight-week FR, and atrophy observed in several organs after four-week FR disappeared or were attenuated after eight-week FR. In contrast, several parameters showed reverse changes between four- and eight-week FR (T-cho in males and GLDH in females), and increased GGT in males and decreased K in females appeared after eight-week FR.

When suppressed body weight gain and/or decreased food consumption, which are major adverse effects in toxicity studies, are noticed in juvenile toxicity studies, the results of this study would be helpful in distinguishing not only between direct effects of the test compounds and secondary effects of malnutrition but also age-dependent compound effects from malnutrition effects.

ACKNOWLEDGMENTS

We thank Shin Nippon Biomedical Laboratories, Ltd. for conducting the animal study, and Dr. Keiichiro Sato for his careful support in study planning.

Conflict of interest

The authors declare that there is no conflict of interest.

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