Hiroshi Sumida

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.

Formal genesis of the outflow tracts of the heart revisited: previous works in the light of recent observations.

The formal genesis of the great arteries continues to be controversial due to the lack of consensus of septation of the developing outflow tract. In order to make it clear how the great arteries are generated, we have re‐examined our previous papers which emphasized the formation of the aorta and pulmonary trunk, concept of the aorticopulmonary septum, formation of the leaflets of semilunar valves, morphogenesis of the crista supraventricularis, programmed cell death and rotation of the outflow tract. In the present paper, we compare outcomes gained from the re‐examination of our previous papers with prevalent interpretations of the arterial trunk. We obtained conclusions as follows: (i) The elongation of the fourth and sixth aortic arch arteries, which sprout from the wall of the aortic sac at the expense of the distal truncus, contributes to the formation of the aorta and pulmonary trunk; (ii) Smooth muscle cells of the tunica media of the arterial trunks do not arise from the transformation of the myocardial cells of the truncus wall (not ‘arterialization’); (iii) Truncus swellings are divided into two parts: distal and proximal. The former contributes to the separation of the orifices of arterial trunks (‘aorticopulmonary septum’). The latter contributes to the formation of the leaflets of the semilunar valves of the aorta and pulmonary trunk; (iv) The origin of the myocardial cells of the crista supraventricularis is a wall of the conus originated from secondary/anterior heart fields; and (v) There has been no acceptable proof that rotation and counterclockwise rotation are involved.

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.

Effect of BCL-2 down-regulation on cellular life span

Reactive oxygen species (ROS) are toxic for cells. BCL-2 is known as the anti-death protein and acts as an antioxidant. When theBCL-2 level of normal fibroblasts was suppressed by antisense bcl-2oligodeoxynucleotide or antisense bcl-2 RNA expression, the life span of the culture was shortened by about 11 population doublings(approx. 15% of the total life span) in comparison to the control culture. Since about twice as many cell deaths were observed in the antisense culture than in the vector culture, the life span shortening was probably caused by ROS-induced death. Acceleration of telomere shortening was not evident in the antisense culture. Other BCL-2 family proteins showed no significant change in expression. Cell death was suppressed by N-acetyl-L-cysteine, an antioxidant, suggesting that ROS were the major cause of cell death. In conclusion, reduction of BCL-2 makes cells more sensitive to death induced by ROS and leads to shortening of the cultures lifespan.

Life span shortening of normal fibroblasts by overexpression of BCL-2: a result of potent increase in cell death

It is well known that BCL-2 protects against cell death by both apoptosis and necrosis. The culture of bcl-2-transfected normal fibroblasts showed a shorter life span by about 12 population doubling levels compared to that of vector transfectants (64 vs 76 population doubling levels, respectively). An MTT assay revealed that BCL-2-overexpressing cells (HCA2/bcl-2) showed more severe growth suppression due to hydrogen peroxide or doxorubicin treatment than vector control cells (HCA2/vector). We observed a significant number of dead cells in the HCA2/bcl-2 culture, but not in the HCA2/vector culture. Other BCL-2 family proteins with both antiapoptotic and proapoptotic activity and other apoptosis-related factors were maintained at similar levels, indicating that overexpression of BCL-2 is the major reason that normal fibroblasts are sensitized to cell death. A broad caspase inhibitor (z-Val-Ala-Asp-fmk) and inhibitors of specific caspases (acetyl-Asp-Glu-Val-Asp-CHO, acetyl-Ile-Glu-Thr-Asp-CHO, and acetyl-Leu-Glu-His-Asp-CHO) suppressed cell death of HCA2/bcl-2 effectively, suggesting involvement of caspase 3-, 8-, and 9-dependent pathways in cell death and that the form of death is apoptosis. Unexpectedly, involvement of active MEK in cell death was shown by the use of its inhibitor, suggesting that crosstalk between BCL-2 and the MAP kinase cascade regulates death as well as life span.

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.