Chemical-induced lung tumor in Tg-rasH2 mice: a novel mouse tumor model to assess immune checkpoint inhibitors combined with a chemotherapy drug.

In subcutaneous tumor models, changes in the tumor microenvironment can lead to differences in therapeutic treatment responses between the subcutaneous and parent tumors. Accordingly, we generated a lung carcinogenesis model that combines genetically modified mice (Tg-rasH2 mice) with two-stage chemical carcinogenesis as an alternative to the subcutaneous tumor model. In this model, Tg-rasH2 mice were treated with 1-ethyl-1-nitrosourea, followed by butylhydroxytoluene. Mice developed lung adenomas five weeks after treatment initiation. Subsequently, anti-mouse PD-1 antibody (α-mPD-1) or isotype control was administered intraperitoneally twice a week for 4 weeks. Tumor growth was examined by measuring the relative tumor area in serially sliced lung histopathological specimens. No statistically significant differences were observed in the relative lung tumor areas between treated and control groups. A second experiment then examined the antitumor efficacy of α-mPD-1 combined with gemcitabine in a mouse model. Mice were treated identically as in Experiment 1, except that the treated group received once-weekly intraperitoneal injections of 10 mg/kg gemcitabine. In contrast to Experiment 1, the combined treatment significantly reduced the relative tumor areas in the lungs. This result also resembles that of a phase III clinical trial (ORIENT-12), showing that patients with non-small-cell lung carcinoma benefited from combination treatment with gemcitabine and the anti-human PD-1 antibody sintilimab. Thus, this mouse model could be a feasible means to preclinically evaluate the antitumor efficacy of different immunotherapy and chemotherapy drug combinations.

Characterization of 5-Fluorouracil Daily Oral Dosing versus Dietary Restriction on Femoral Growth Plates in Rats.

We evaluated the growth plates (GPs) of rats after a 14-day reduction in food consumption caused by either daily oral dosing with 5-fluorouracil (5-FU: a positive control reducing food consumption and affecting the GPs) or a direct reduction in food consumption to determine whether the observed changes were attributable to a direct effect of drug toxicity. Histomorphometric analyses of the femoral GP were performed for a nontreated (NT) control group, three groups treated with 5-FU (12, 15, and 18 mg/kg/day) and three groups with food intake restricted to levels corresponding to those consumed by the rats in the three 5-FU-treated groups. Compared with the NT group, the GP widths and the number of chondrocytes in the proliferative zone decreased significantly in all the 5-FU-treated groups and the dietary restriction groups. Importantly, no significant differences between the 5-FU-treated groups and the groups with matched dietary restrictions were seen for most parameters. Thus, the 14-day dietary restriction caused significant changes in the proliferative zone of the GP, and similar changes observed in the 5-FU-treated groups were presumed to result from the comparable reduction in food intake rather than being a direct toxic effect of the drug.

Feature on erythropoiesis in dietary restricted rats.

We attempted to characterize the influence of undernutrition on erythropoiesis in toxicity studies. Male rats were divided into the following 5 groups: dietary restriction groups in which feeding was restricted by 33% or 66% for 14 days (R33 and R66); phlebotomy groups in which 1% or 4% of total blood volume was removed by serial phlebotomy for 14 days (PB01 and PB04); and a nontreated group (NT). Toxicological parameters such as hematology and blood chemistry were evaluated. The body weight gains in the R33 and phlebotomy groups (PB01 and PB04) were similar and were less than that observed in the NT group. Decreases in peripheral blood reticulocytes, bone marrow erythroids and the unsaturated iron binding capacity (UIBC) were observed as changes that suppressed erythropoiesis in the R33 and R66 groups. However, increases in reticulocytes and UIBC were observed as opposite changes in the phlebotomy groups. In addition, an increase in the blood urea nitrogen level and a decrease in the serum alkaline phosphatase level were observed as changes reflecting poor nutrition in the phlebotomy groups. Decreased reticulocytes which are related to poor nutrition were not observed. However, increases in those cells as reflected by a loss of blood were observed in the phlebotomy groups. Even if undernutrition suppresses erythropoiesis, the ability of erythropoiesis to respond to a demand appears to be retained. In repeated dose toxicity studies, decreased food consumption is often observed in the drug administration groups. Our study results provide useful information for hematological evaluations in toxicity studies.

Full-field electroretinography obtained using a contact lens electrode with built-in high-intensity white-light-emitting diodes can be utilized in toxicological assessments in rats.

Full-field electroretinography (ERG) using contact lenses with built-in LED was performed on albino rats, and used to evaluate the visual toxicity of sodium iodate (NaIO(3)). Experiment 1 was carried out to determine the optimal conditions in rats relating to stimulus intensity, background illumination, and light adaptation period. As a result, we found that a full-field ERG was recorded under the following conditions: stimulus intensity: -3.5 log cd s/m(2) in rod response; background intensity and light adaptation period: 10 cd/m(2) and 10 min in cone and flicker responses. Experiment 2 was carried out to confirm the usefulness of full-field ERG using rats with retinal toxicities induced by NaIO(3). Male rats were given NaIO(3) intravenously at a dose of 50 mg/kg. ERG was recorded before administration and after 3, 8, 24 h, and 7 days of administration, and histopathological analysis was conducted after 8 h of administration. The rod response disappeared completely at 3 h, based on a reduced maximal response and oscillatory potentials. On the other hand, cone and flicker responses were still present at 8 h. All responses disappeared on the 7th day. These findings indicate that the retinal toxicity induced by NaIO(3) was expressed first in rods, followed by cones. There were no microscopical changes after 8 h of administration, although the rod responses had completely disappeared by this time. These results suggest that full-field ERG in rats using an LED contact lens is useful for the separate evaluation of toxic effects on rods and cones.

[Circadian rhythms and effects of anesthesia on plasma natriuretic peptide levels in rats].

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are circulating hormones secreted predominantly in patients with hypertension or congestive heart failure. To obtain background data on plasma ANP and BNP levels in rats, we investigated the circadian rhythms and effects of anesthesia on these peptides. To determine the circadian rhythms, plasma samples from thirty rats were collected by non-anesthesia (decapitation) at six time points every fourth hour. To determine the effects of anesthesia, plasma samples from thirty-two rats were collected under diethyl ether, pentobarbital or urethane anesthesia. The plasma ANP and BNP levels were determined using a radioimmunoassay. The plasma ANP levels were high from the evening to early morning, while the plasma BNP levels were relatively low at 2:30 AM. The difference in the BNP levels was statistically significant. The plasma BNP levels were relatively high when the rats were anesthetized using urethane. These results suggest that blood collection should be performed between 10:30 AM to 2:30 PM to determine plasma ANP and BNP. The use of pentobarbital is also recommended for toxicological studies in rats.

Evaluation of myelotoxicity in dietary restricted rats.

The purpose of this study was to clarify the effect of decreased food consumption on evaluation of myelotoxicity in routine general toxicity studies. Male rats were divided into the following 7 groups: 12, 15, and 18 mg/kg 5-fluorouracil (5-FU) treatment groups (FU12, FU15 and FU18); dietary restriction groups (R12, R15 and R18 receiving the same amount of food as the rats in the FU12, FU15 and FU18 groups, respectively); and a nontreated control group (NT). We compared the changes in body weight, hematology and the results of cytological analyses of bone marrow and histopathology among the groups after administration and recovery periods of 14 and 7 days, respectively. At the end of the administration period, the FU15 and FU18 groups showed decreases in many hematologic and bone marrow parameters that were all similar to those in the corresponding dietary restriction groups (R15 and R18). A granulocyte abnormality (polyploidy: frequency of 1% or less) was also observed in all 5-FU treated groups. At the end of the recovery period, increases in the reticulocyte and platelet counts and extramedullary hematopoiesis of the spleen were observed in the 5-FU treated groups. These results indicate that the results of general toxicity studies in rats should be evaluated in consideration of dietary restriction effects when food consumption is decreased at about 30-40% or more. Careful morphological observation of hemocytes would be helpful in distinguishing the effect of a drug from that of dietary restriction in relation to hematological and bone marrow parameters. Performance of a recovery test to determine the reactive response of hematopoiesis is also recommended.

Importance of starting age for myelotoxicity study in dietary restricted rats.

The aim of this study was to prove our hypothesis that adult rats with lowering of body weight gain, rats at 12 weeks of age as an example, are suitable for evaluation of myelotoxicity. Age-related differences between young rats (6-week-old study) and adult rats (12-week-old study) were analyzed in hematological examination values. The data of the young rats were reprinted from our previous report (Miyata et al., 2009) since our hypothesis was verified by comparison with that previous report. Several experimental groups were defined for the 12-week-old study as well as for the 6-week-old study; these included 5-fluorouracil (5-FU) treated groups receiving 12, 15 and 18 mg/kg/day (FU12, FU15 and FU18), pair-feeding groups (R12, R15 and R18 receiving the same amount of food as in the FU12, FU15 and FU18 groups, respectively) and a nontreated control group. Numerous hematologic and bone marrow parameters in the 5-FU treated groups were comparable to those in the corresponding pair-feeding groups in both age studies. Generally, the influences of undernutrition were more apparent in the young rats than in the adult rats. Histopathological examinations showed a decrease in hematopoiesis in the bone marrow in the 5-FU treated and pair-feeding groups. No apparent differences were observed in the decreased hematopoiesis between the 5-FU treated and pair-feeding groups in the 6-week-old study, but a difference between these groups was noted in the 12-week-old study; decreased hematopoiesis was more frequently noted in the 5-FU treated groups. These facts suggest that adult rats are more suitable than young rats for evaluation of 5-FU-induced myelotoxicity.

P-VECP can reveal visual toxicity in pigmented rats of repeated doses of ethambutol.

We determined if pattern visually evoked cortical potentials (P-VECPs) in pigmented rats would reveal visual toxicity induced by a drug even when in cases of repeated doses. We obtained appropriate conditions of P-VECPs measurement; the spatial frequency, 0.16 cycle/degree; the mean stimulation luminance, 25 cd/m(2); and the stimulation frequency, 2 Hz. Twelve adult male pigmented rats (Iar: Long-Evans), weighing 210-301 g, were grouped into two (six per group): the control and the ethambutol (EB) 500 mg/kg administered groups. In the EB 500 mg/kg group, the rats were administered EB subcutaneously once daily for 6 weeks. Rats in the control group were given the vehicle subcutaneously once daily for 6 weeks. P-VECPs were carried out prior to initiation of drug administration and at the first, second, third, fourth, and sixth week of the administration. Prolongation of P1 latency in the P-VECPs was evident in the EB 500 mg/kg at fourth and sixth weeks, and there were no marked changes in the control group and no marked changes in P1N1 amplitude in either group. These findings suggest that P-VECPs in pigmented rats can detect chemically induced visual toxicity even in cases of repeated dosing of a drug. This approach is useful for evaluating the visual toxicity of drugs given repeatedly.

Evaluation of bone toxicity in various bones of aged rats.

The aim of the present study was to provide a method for evaluating bone toxicity induced by drugs in various bones in aged rats. Male Crl:CD (SD) rats at 46 weeks of age were administered 15 mg/m(2) body surface area of doxorubicin, which effects the growth plate in weanling rats, weekly for 9 weeks by intravenous injection, and the femur, sternum, humerus and tibia were examined histopathologically. In the doxorubicin-treated group, thinning of the growth plate was remarkably observed in the proximal tibia and humerus; however, these changes were not observed in other regions. In addition, the osteoclast number per bone perimeter in the proximal tibia was significantly higher than others in control aged rat. Thus, recognizing the various histological reactions related to the time of epiphyseal closure is important for evaluating bone toxicity in aged rats.

Evaluation of Short-term Myelotoxicity Study in Dietary Reduced Rats.

This study attempted to prove our hypothesis that a short-term toxicity study, using a 4-day dosing regimen as an example, is suitable for evaluating myelotoxicity in rats. We compared the hematological, bone marrow cytological and histopathological results of 5-fluorouracil (5-FU) treated and pair-feeding groups after a 4-day administration period. Several experimental groups were defined for this 4-day study as well as for our previously reported 14-day study (Miyata et al., 2009); these included 5-FU treated groups receiving 12, 15 and 18 mg/kg/day (FU12, FU15 and FU18), pair-feeding groups (R12, R15 and R18 receiving the same amount of food as the FU12, FU15 and FU18 groups, respectively) and a nontreated control group. Although severe reductions in body weight gain and food consumption were reported in the 14-day study, only slight reductions were observed in the 4-day study. In the 4-day study, a decrease in blood reticulocytes and a decreasing trend of marrow erythroid cells were only observed in the FU18 group, and no effects were observed in the pair-feeding groups. The erythroblastic changes observed in this 4-day study were thought to reflect the direct influence of 5-FU administration. Since concerns regarding the influence of secondary changes related to undernutrition were minimized in the 4-day study, it was thought to clarify the direct influence of 5-FU administration on erythroblastic cells. Thus, a 4-day study protocol might be helpful for distinguishing secondary changes related to undernutrition.