Heat shock during rat embryo development in vitro results in decreased mitosis and abundant cell death.

Epidemiologic studies strongly suggest that in utero exposure to hyperthermia results in developmental defects in humans. Rats, mice, guinea pigs, and other species exposed to hyperthermia also exhibit a variety of developmental defects. Studies in our laboratory have focused on exposure to hyperthermia on Gestation Day (GD) 10 of rats in vivo or in vitro. Within 24 h after in vivo or in vitro exposure, delayed or abnormal CNS, optic cup, somite, and limb development can be observed. At birth, only rib and vertebral malformations are seen after hyperthermia on GD 10, and these have been shown to be due to alterations in somite segmentation. Unsegmented somites have been thought to result from a cell-cycle block in the presomitic mesoderm, from which somites emerge individually during normal development. In the present study, DNA fragmentation (terminal deoxynucleotidyl transferase (TdT) catalyzed fluorescein-12-dUTP DNA end-labelling), indicative of apoptotic cell death, and changes in cell proliferation were examined in vitro in 37 degrees C control and heat treated (42 degrees C for 15 min) GD 10 CD rat embryos. Embryos were returned to 37 degrees C culture following exposure and evaluated 5, 8, or 18 h later. A temperature-related increase in TdT labelled cells was observed in the CNS, optic vesicle, neural tube, and somites. Increased cell death in the presomitic mesoderm also was evident. Changes in cell proliferation were examined using the cell-specific abundance of proliferating cell nuclear antigen (PCNA) and the quantification of mitotic figures. In neuroectodermal cells in the region of the optic cup, a change in the abundance of PCNA was not apparent, but a marked decrease in mitotic figures was observed. A significant change in cell proliferation in somites was not detected by either method. These results suggest that acute hyperthermia disrupts embryonic development through a combination of inappropriate cell death and/or altered cell proliferation in discrete regions of the developing rat embryo. Furthermore, postnatal vertebral and rib defects following disrupted somite development may be due, in part, to abundant cell death occurring in the presomitic mesoderm.

Evaluation of octamethylcyclotetrasiloxane (D4) as an inducer of rat hepatic microsomal cytochrome P450, UDP-glucuronosyltransferase, and epoxide hydrolase: a 28-day inhalation study.

Repeated inhalation exposure to octamethylcyclotetrasiloxane (D4) produces a reversible and dose-related hepatomegaly and proliferation of hepatic endoplasmic reticulum in rats. However, the effects of D4 on the expression of cytochrome P450 enzymes have not been evaluated. In the present study, the time course for changes in hepatic microsomal cytochrome P450 enzyme expression following repeated inhalation exposure to D4 vapors was determined in male and female Fischer 344 rats. Animals were exposed to D4 vapor at concentrations of 70 and 700 ppm, via whole body inhalation for 6 h/day, 5 days/week for 4 weeks. Specified animals were euthanized on exposure days 3, 7, 14, 21, and 28. Microsomal fractions were prepared from fresh liver by differential centrifugation. Enzyme activity as well as immunoreactive protein levels of several cytochrome P450 enzymes (CYP), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were evaluated. The time course for enzyme induction was monitored by measuring 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD) activities on days 3, 7, 14, 21, and 28. CYP1A1/2 activity, as determined by EROD activity, was increased approximately 2- to 3-fold over the exposure period. However, an examination of immunoreactive protein revealed no induction of CYP1A1 and a suppression of CYP1A2 in the 700 ppm D4 group. In comparison, CYP2B1/2 enzyme activity, as determined by PROD, was significantly increased as early as day 3 in both the 70 and 700 ppm D4 groups of male and female rats. Overall, PROD activity on day 28 was induced more than 10-fold in the 70 ppm D4 groups and more than 20-fold in the 700 ppm D4 groups. The increase in PROD activity was paralleled by a comparable increase in CYP2B1/2 immunoreactive protein. There was a modest (2- to 3-fold) increase in CYP3A1/2 activity and immunoreactive protein, as determined by 6 beta-hydroxylation of testosterone and Western blot analysis. Expression of CYP enzymes was at or near maximum by day 14 and remained relatively constant throughout the exposure period. On day 28, epoxide hydrolase activity and immunoreactive protein were induced (2- to 3-fold) in a dose-dependent manner. Only slight changes in the expression and activity of UDPGT were detected, and these did not appear to be dose related. Thus, repeated inhalation exposure to D4 induces CYP enzymes and epoxide hydrolase in a manner similar to that observed for phenobarbital (PB). Therefore, D4 can be described as a "PB-like" inducer of hepatic microsomal enzymes in the Fischer 344 rat.

Cellular adaptation to chronic cadmium exposure: intracellular localization of metallothionein protein in human trophoblast cells (JAr).

Trophoblast cells are the first embryonic cells that modulate the transfer of a variety of compounds (oxygen, amino acids, xenobiotics, metals) from the maternal to the fetal circulation in the human placenta. Human placental exposure to the toxic metal, cadmium (Cd) results in a decrease in the production of human chorionic gonadotropin (hCG), a decrease in the maternal to fetal transport of zinc (Zn), and trophoblastic necrosis. Thus, the ability of trophoblast cells to adapt to exposure to the toxic metal Cd has been considered crucial. In this study, the expression and intracellular localization of metallothionein (MT), a small molecular weight, metal binding protein, was examined in trophoblast cells (JAr) grown in normal media and in cells exposed chronically (6 months) to 2 microM CdCl2. Conventional and confocal fluorescence microscopy were used to examine the intracellular localization of MT protein in control cells and cells grown chronically in Cd. In unexposed trophoblast cells, MT protein was primarily perinuclear with low level, punctate expression in the cytosol. Following both chronic and 24 hour exposure to Cd, MT protein levels were increased (at least 3-fold in both chronic and acute exposures) and the protein was now concentrated inside the nucleus with a lacy, cytoskeletal pattern of expression in the cytosol. To determine if the nuclear accumulation of MT protein was dependent on new protein synthesis, control cells were exposed to CdCl2 (2 microM) and cycloheximide (2 micrograms/. ml) for 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Human placenta makes extracellular glutathione peroxidase and secretes it into maternal circulation.

Extracellular glutathione peroxidase (eGPX) is a selenoglycoprotein distinct from cellular glutathione peroxidase (cGPX). The cDNA for eGPX has recently been cloned from human placenta. To determine whether human placenta makes both cGPX and eGPX and secretes eGPX, we used specific immunoprecipitations of 75Se metabolically labeled proteins from full-term placental explants in culture and perfused placental lobules. Placental explants and metabolically active, dually perfused placental lobules synthesized and contained both cGPX and eGPX and secreted eGPX. Perfused tissue secreted eGPX into the maternal but not into the fetal perfusate. In situ hybridizations using antisense and sense eGPX riboprobes were performed on sections of first-, second-, and third-trimester placentas. In the first-trimester placenta, transcripts were localized predominantly to cytotrophoblast cells, whereas in the full-term placenta syncytiotrophoblast cells and stromal cells but not fetal endothelial cells expressed eGPX mRNA. It is concluded that human placenta synthesizes both cGPX and eGPX and secretes eGPX into the maternal circulation, consistent with the location of the eGPX mRNA.

Cell-specific increases in metallothionein expression in the human placenta perfused with cadmium.

Metallothionein (MT) is a cysteine-rich protein that may have both a nutritional and a protective role by binding either physiologic or toxic concentrations of metals in cells. The objective of this investigation was to measure the inducibility of MT mRNA and protein, and to determine their specific cellular localization following exposure to 20 microM cadmium (Cd) in the perfused human placenta for periods up to 8 h. MT mRNA was quantitated using slot blot hybridization with an MT IIa genomic clone and MT transcripts were localized via in situ hybridization. MT protein was measured using a 109Cd-binding assay and localized by immunocytochemistry using a monoclonal antibody to MT in fresh placental tissue from nonsmoking mothers and in tissue perfused for 4 or 8 h with CdCl2 (20 microM). Perfusions were performed on placentae from which fresh samples were also obtained. In fresh term placentae, MT mRNA was barely detectable using both slot blot and in situ hybridization. Slot blot hybridization for MT message demonstrated a dramatic increase of at least 70-fold above fresh after 8 h of perfusion with 20 microM CdCl2. In this time frame, however, statistically significant increases in MT protein were not detected. Following perfusion for 4 and 8 h with 20 microM CdCl2, accumulation of MT transcripts was shown, in situ, to occur in stromal and endothelial cells with a small but detectable increase in trophoblast cells. These results were consistent with the localization of the MT protein after perfusion with Cd and also correlated with areas of stromal edema. Thus, while the trophoblast cells are in direct contact with Cd in maternal perfusate, the cells of the villous core and fetal endothelial cells are most responsive to Cd exposure with regard to MT expression. Lower levels of MT expression in trophoblast cells may leave this cell type more susceptible to Cd toxicity, while higher levels of MT expression in cells of the villous core and endothelial cells may provide these cells with more protection.

Andegerebenha vowel phonology.

The most common vowel system in Australian languages and the system ancestral to other Australian vowel systems is the triangular system /i-u-a/, with or without phonemic length. This paper shows that this is not appropriate for Andegerebenha, considers the suitability of a two-vowel system /e-a/, and rejects this in favour of one vowel plus length /a-a-/. The short vowel is greatly influenced in quality by the adjacent consonants; the longer one much less so. The consonant inventory is augmented by the addition of a number of labialised consonants, which embody the rounding of earlier *u. Conversely, the phenome/V/, a velar glide, was introduced when an earlier *w acquired the feature [-round] of adjacent vowels.