Akihiro Arima

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects bone tissue in rhesus monkeys.

Bone tissue is one of the target tissues for dioxins and dioxin-like compounds. Therefore, the aim of this study was to investigate effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on bone tissue in rhesus monkey, the most human-like experimental model available. Pregnant rhesus monkeys (Macaca mulatta; age 4-10 years) were exposed to TCDD with a total dose of 40.5-42.0 or 405-420ng/kg bodyweight by repeated subcutaneous injections starting at gestational day 20 and followed by injections every 30 days until 90 days after delivery. At a mean age of 7 years the offspring were sacrificed and the femur bone dissected. Results from peripheral Quantitative Computed Tomography (pQCT) analyses of the metaphyseal part of the femur bones in female offspring showed significant increases in trabecular bone mineral content (BMC; +84.6%, p<0.05, F-value (F)=5.9) in the low-dose treatment group compared with the controls. In the same animals, analysis of the mid-diaphyseal part revealed increases in total BMC (+21.3%, p<0.05, F=5.2) and cortical cross-sectional area (CSA; +16.4%, p<0.01, F=7.4) compared with the controls. In males, changes in biomechanical properties indicating more fragile bone were observed. Displacement at failure were significantly increased in the male low-dose group compared to the controls (+38.0%, p<0.05, F=11). The high dose of TCDD did not induce any significant changes in bone morphology. In conclusion, in utero and lactational low-dose, but not high-dose exposure to 2,3,7,8-TCDD induced disruption of bone tissue development in rhesus monkey, a result suggesting that similar effects might occur in humans also.

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces a reduction in epididymal and ejaculated sperm number in rhesus monkeys

A long-term developmental toxicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure was performed in rhesus monkeys and the effect on male reproductive organs was determined in the second generation. Dams received 0, 30 or 300 ng/kg TCDD subcutaneously on Day 20 of gestation, and then 5% of the initial dose was injected every 30 days until Day 90 after delivery. The offspring were maintained until reaching sexual maturity, and evaluated by semen analysis, and histopathology of the testes and epididymides. Ejaculated sperm concentration was severely reduced at 300 ng/kg, and sperm viability and activity were dose-proportionally reduced, although effects on spermatogenesis were slight. Histomorphometry revealed markedly reduced area of the ductus epididymis accompanying decreased reserved sperm in the 30 and 300 ng/kg groups. In conclusion, in utero and lactational exposure to TCDD induced a reduction of sperm quality in rhesus monkeys.

Developmental toxicity of dibutyltin dichloride given on three consecutive days during organogenesis in cynomolgus monkeys

We previously reported that the administration of dibutyltin dichloride (DBTCl) by nasogastric intubation during the entire period of organogenesis, days 20–50 of pregnancy, was embryolethal, but not teratogenic, in cynomolgus monkeys. The present study was conducted to further evaluate the developmental toxicity of DBTCl given to pregnant monkeys on 3 consecutive days during organogenesis. Cynomolgus monkeys were given DBTCl at 7.5 mg/kg body weight/day by nasogastric intubation on days 19–21, 21–23, 24–26, 26–28, 29–31, 31–33, or 34–36 of pregnancy, and the pregnancy outcome was determined on day 100 of pregnancy. Embryonic/fetal loss was observed in 1 female given DBTCl on days 19–21, 2 females given DBTCl on days 24–26, and 1 female given DBTCl on days 34–36. There were no effects of DBTCl on developmental parameters in surviving fetuses, including fetal body weight, crown-rump length, tail length, or placental weight. No external, internal, or skeletal malformations were detected in fetuses in any group. DBTCl did not affect the incidence of fetuses with skeletal variation or skeletal ossification of fetuses. These data confirm our previous findings that DBTCl was embryolethal, but not teratogenic, in cynomolgus monkeys.

Historical control data on prenatal developmental toxicity studies in rabbits

Historical control data on rabbit prenatal developmental toxicity studies, performed between 1994–2010, were obtained from 20 laboratories, including 11 pharmaceutical and chemical companies and nine contract laboratories, in Japan. In this paper, data were incorporated from a laboratory if the information was based on 10 studies or more. Japanese White rabbits and New Zealand White rabbits were used for prenatal developmental toxicity studies. The data included maternal reproductive findings at terminal cesarean sections and fetal findings including spontaneous incidences of morphological alterations. No noticeable differences between strains or laboratories were observed in the maternal reproductive and fetal developmental data. The inter‐laboratory variations in the incidences of fetal external, visceral, and skeletal alterations seem to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, and terminology of fetal alterations.

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces disruption of glands of the prostate and fibrosis in rhesus monkeys.

We investigated the effects that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure has on the prostate in rhesus monkey offspring. Dams received 0, 30 or 300 ng/kg TCDD subcutaneously on Day 20 of gestation, and then 5% of the initial dose was injected every 30 days until Day 90 after delivery. The offspring were maintained until reaching sexual maturity, and examined histopathologically. Dose-dependent decreases in glands of the prostate and widespread fibrosis were observed in offspring. It is noteworthy that 7 years from the final lactational TCDD exposure, inflammatory cell infiltration and disruption of glands of the prostate were still observed. Differential mRNA expression associated with fibrosis, inflammatory response and disruption of cell components were demonstrated by microarray analysis, with up-regulation of TGM4, TGFB1, COL1A1 and MMP2 confirmed. In conclusion, in utero and lactational exposure to TCDD induced dose-related prostatic fibrosis, indicating prostatic dysfunction and inducible semen quality reduction in second-generation rhesus monkeys.

Fetal and neonatal goiter in cynomolgus monkeys following administration of the antithyroid drug thiamazole at high doses to dams during pregnancy.

To evaluate morphologic alterations in the thyroid gland in the second generation in cynomolgus monkeys, pregnant dams were exposed to high doses of thiamazole. In Experiment A, dams received thiamazole intragastrically via a nasogastric catheter from gestation day (GD) 50 to GD 150 or on the day before delivery. Initially, the dose level was 20 mg/kg/day (10 mg/kg twice daily); however, the dose level was subsequently decreased to 5 mg/kg/day (2.5 mg/kg twice daily), since deteriorated general conditions were observed in two dams. Six out of seven neonates died on the day of birth. The cause of neonatal death was tracheal compression and suffocation from goiter. The transplacental exposure to thiamazole affected the fetal thyroid glands and induced goiter in all neonates. The surviving neonate was necropsied 767 days after discontinuation of thiamazole exposure and showed reversibility of the induced changes. In Experiment B, dams were intragastrically administered thiamazole at 5 mg/kg/day (2.5 mg/kg twice daily) for treatment periods from GDs 51 to 70, 71 to 90, 91 to 110, 111 to 130 and 131 to 150. All fetuses showed enlarged thyroid glands but were viable. Histopathologically, hypertrophy and/or hyperplastic appearance of the follicular epithelium of the thyroid gland was observed at the end of each treatment period. The most active appearance of the follicular epithelium, consisting of crowded pedunculated structure, was demonstrated at end of the treatment period from GD 131 to 150. This is the first report on the morphology of fetal and neonatal goiter in the cynomolgus monkey.

Placental and Fetal Effects of Onartuzumab, a Met/HGF Signaling Antagonist, When Administered to Pregnant Cynomolgus Monkeys

Onartuzumab is an engineered single arm, monovalent monoclonal antibody that targets the MET receptor and prevents hepatocyte growth factor (HGF) signaling. Knockout mice have clearly demonstrated that HGF/MET signaling is developmentally critical. A pre- and postnatal development study (enhanced design) was conducted in cynomolgus monkeys to evaluate the potential developmental consequences following onartuzumab administration. Control or onartuzumab, at loading/maintenance doses of 75/50 mg/kg (low) or 100/100 mg/kg (high), was administered intravenously once weekly to 12 confirmed pregnant female cynomolgus monkeys per group from gestation day (GD) 20 through GD 174. Onartuzumab administration resulted in decreased gestation length, decreased birth weight, and increased fetal and perinatal mortality. A GD147 C-section was conducted for a subset of Control and High Dose monkeys, and identified placental infarcts with hemorrhage in the chorionic plate, chorionic villus and/or decidual plate. These findings were limited to placentas from onartuzumab-treated animals. In addition, decreased cellularity of the hepatocytes with dilated hepatic sinusoids was inconsistently observed in the liver of a few fetal or infant monkeys that died in the perinatal period. Surviving offspring had some evidence of developmental delay compared with controls, but no overt teratogenicity. Overall, effects on the perinatal fetuses were consistent with those reported in knockout mice, but not as severe. Onartuzumab concentrations were low or below the level of detection in most offspring, with cord blood concentrations only 1%-2% of maternal levels on GD 147. Malperfusion secondary to onartuzumab-induced placental injury could explain the adverse pregnancy outcomes, fetal growth restriction and relatively low fetal exposures.

Assessment of placental transfer and the effect on embryo-fetal development of a humanized monoclonal antibody targeting lymphotoxin-alpha in non-human primates.

An enhanced embryo-fetal development study was conducted in cynomolgus monkeys using pateclizumab, a humanized IgG1 monoclonal antibody (mAb) targeting lymphotoxin-alpha. Pateclizumab administration between gestation days (GD) 20 and 132 did not induce maternal or developmental toxicities. The ratio of fetal-to-maternal serum concentration of pateclizumab was 0.73% on GD 50 and 61% by GD 139. Decreased fetal inguinal lymph node-to-body weight ratio was present in the high-dose group without microscopic abnormalities, a change attributable to inhibition of lymphocyte recruitment, which is a pharmacologic effect of pateclizumab during late lymph node development. The effect was observed in inguinal but not submandibular or mesenteric lymph nodes; this was attributed to differential susceptibility related to sequential lymph node development. Placental transfer of therapeutic IgG1 antibodies; thus, begins during the first trimester in non-human primates. Depending on the potency and dose levels administered, antibody levels in the fetus may be pharmacologically or toxicologically relevant.

Utility of finger maze test for learning and memory abilities in infants of cynomolgus monkeys exposed to thiamazole

A new type of learning and memory test using a finger maze was conducted in infant cynomolgus monkeys that were exposed to thiamazole (2 and 3.5 mg/kg per day to pregnant animals orally) during the fetal period (gestational days 120 to 150). We modified Tsuchidas original finger maze test method by reducing the number of trials per day and simplifying the criteria for achievement of training, and we added a long‐term memory test. In the memory test, thiamazole‐exposed infants required greater time to complete the finger maze test than the control infants although no effect was noted in the training or learning test. The results suggest that an impaired long‐term memory could be detected by our modified finger maze test.

Effects of maternal exposure to thiamazole on behavioral development in infant cynomolgus monkeys

Thiamazole, an anti‐hyperthyroidism agent, was administered orally to pregnant cynomolgus monkeys at doses of 2.0 and 3.5 mg/kg per day from GD 120 to GD 150 to investigate effects on behavioral development of their infants. Swelling of the throat region due to enlargement of the thyroid glands was observed at birth in thiamazole‐treated infants, and it returned to normal around postnatal day (PND) 30. At necropsy of infants at 12 months of age, thyroidal weight in the thiamazole groups was increased. This finding suggested the likelihood that administration of thiamazole to maternal animals during the late gestational period induced thyroid goiter in fetal/infant monkeys through placental transfer of thiamazole. No clear changes were noted in thyroid histopathology or serum thyroid hormone levels in maternal animals or infants, but goiter formation might have been indicative of exposure to high thyroid stimulating hormone (TSH) and low T3 or T4 in utero from maternal treatment with thiamazole. Age‐related changes were observed in the control in behavioral development tests, while infants at 3.5 mg/kg showed no age‐related decrease in contact behavior and no increase in exploratory activity on PND 90 or PND 170. In addition, the number of eye contacts between PND 210 and PND 240 was less frequent. This indicated that maternal exposure to thiamazole induced mental retardation‐like behaviors in infants. Thiamazole may directly inhibit thyroid hormone synthesis in the fetus by placental transfer. From these results, it was speculated that oral administration of thiamazole to maternal animals during the late gestational period induced retardation of behavioral development in their infants.