IGF-1 Mediates EphrinB1 Activation in Regulating Tertiary Dentin Formation.

Eph receptors belong to a subfamily of receptor tyrosine kinases that are activated by membrane-spanning ligands called ephrins. Previously, we demonstrated that the ephrinB1-EphB2 interaction regulates odontogenic/osteogenic differentiation from dental pulp cells (DPCs) in vitro. The goal of this study was to identify the molecular mechanisms regulated by the EphB2/ephrinB1 system that govern tertiary dentin formation in vitro and in vivo. During tooth development, ephrinB1, and EphB2 were expressed in preodontoblast and odontoblasts at postnatal day 4. EphrinB1 was continuously expressed in odontoblasts and odontoblastic processes until the completion of tooth eruption. In addition, ephrinB1 was expressed in odontoblastic processes 2 wk following tooth injury without pulp exposure, whereas EphB2 was expressed in the center of pulp niches but not odontoblasts. In a model of tooth injury with pulp exposure, ephrinB1 was strongly expressed in odontoblasts 4 wk postinjury. In vitro studies with human and mouse DPCs treated with calcium hydroxide (CH) or mineral trioxide aggregate (MTA) showed an increased expression of insulin-like growth factor 1 (IGF-1). Experiments using several inhibitors of IGF-1 receptor signaling revealed that inhibiting the Ras/Raf-1/MAPK pathway inhibited EphB2 expression, and inhibiting the PI3K/Akt/mTOR pathway specifically inhibited ephrinB1 gene expression. Tooth injury in mice with odontoblast-specific IGF-1 receptor ablation exhibited a reduced tertiary dentin volume, mineral density, and ephrinB1 expression 4 wk following injury. We conclude that the IGF-1/ephrinB1 axis plays significant roles in the early stages of tooth injury. Further research is needed to fully understand the potential of targeting ephrinB1 as a regenerative pulp therapy.

Single-pixel demonstration of innovative adaptive optics by use of a charge-transfer membrane light modulator.

We have constructed and demonstrated a single-pixel implementation of an all-optical membrane-based spatial light modulator as a compact optical wave-front error correction device. High rates of response of as much as 20 kHz in an open-loop configuration were obtained. The device was then used in an adaptive-optics servo to compensate successfully for a 1-kHz sinusoidal phase error with a peak-to-peak excursion of approximately pi/7 rad. A small-signal servo gain of the order of 10 was inferred from the closed-loop measurements.

Charge-transfer-plate spatial light modulators.

Charge-transfer-plate spatial light modulators (CTPSLM's) are a class of devices that employ chargetransfer plates as the interface between the charge-generation element and the light-modulation element. Both optically addressed and electrically addressed devices have been built. Chargegenerating elements for the optically addressed devices include photoconductors, photodiode and phototransistor arrays, optoelectronic integrated circuit chips, and photocathode-microchannel-plate assemblies. For electrically addressed devices, electron guns, very large-scale integrated circuits, thin-film transistors, and matrix electrodes are among the possible charge-generation elements. Lightmodulation elements used in CTPSLM's include liquid crystals, electro-optic organic and inorganic crystals, polymers, deformable membrane mirrors, oil films, multilayer dielectric films, and electroluminescent films. In principle, all combinations of charge-generation elements and light-modulating elements are possible. This paper explores the fundamental performance limitations of CTP technology, and describes the design, operation, and applications of five different CTPSLM's (three based on membrane-mirror technology and two on liquid-crystal technology).

Electron-beam-addressed membrane mirror light modulator for projection display.

The performance of a prototype, reflection-mode projection display based on an electron-beam-addressed membrane mirror light modulator (e-MLM) is described. The e-MLM converts electronic video information into a two-dimensional phase object, that is then schlieren imaged onto a screen. High-contrast dynamic projection of images is demonstrated over a broad range of wavelengths, from the visible to the midinfrared. As such this device is expected to find applications in large-screen visible displays and dynamic infrared scene projectors.

Effects of adenine and its isomer 4-aminopyrazolo-[3,4-d]-pyrimidine on 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced mortality in rats.

Adult male Fischer rats were given a single po dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equal to 2 times the LD50 to increase the serum and liver lipid concentrations and to induce mortality. In addition, animals were given 4-aminopyrazolo-[3,4-d]-pyrimidine (4APP), an agent that decreases serum lipids, or adenine (Ad), an agent that prevents the formation of fatty liver, to examine the relationship between changes in lipids and TCDD-induced mortality. The principal effect of 4APP on TCDD-induced mortality (325 micrograms TCDD/kg body wt) was that it shortened the mean time to death. In contrast, Ad stimulated feed consumption and decreased body weight loss, but the mean times to death were similar for TCDD and TCDD + Ad animals. Based on these mortality studies, 4APP, but not Ad, affects the TCDD-induced mortality in Fischer rats. The TCDD-induced sensitivity to 4APP, based on decreased mean time to death, implies that blocking the release and/or synthesis of triglyceride-rich lipoproteins by the liver, and the subsequent decrease in serum lipids, may play an important role in the TCDD-induced mortality. The increase in serum triglyceride associated with TCDD exposure appears to be essential in providing metabolic energy under circumstances where lipoprotein retrieval is reduced.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and fasting on body weight and lipid parameters in rats.

This study compared the effect of fasting (feed deprivation) and the effect of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on body weight and on key lipid parameters. The time-course study indicated a statistically significant (p less than 0.05) body weight loss in the TCDD-exposed rats 5 days after the oral administration of corn oil with TCDD (60 micrograms/kg body wt). Animals demonstrated a 10% body weight decrease either 1 week after this TCDD administration or after 72 hr of fasting. Marked increases in serum triglyceride and cholesterol were observed only in the TCDD-exposed rats, but not in the 0- or 72-hr fasted control rats. These results indicated that some body weight loss and decreased feed consumption occurred after TCDD exposure, but that the metabolic response, with respect to serum lipid metabolism, was not that of a control rat that had lost a similar amount of body weight due solely to fasting.

Free radicals in the stratosphere: a new observational technique.

A new approach to in situ observations of trace reactive species in the stratosphere is described. A balloon-borne system, floating 40 kilometers above the earth's surface, successfully lowered and then retracted a cluster of instruments a distance of 12 kilometers on a filament of Kevlar. This instrument cluster is capable of detecting gas-phase free radicals at the part-per-trillion level. The suspended instrument array has excellent stability and has been used to measure atomic oxygen concentrations in the stratosphere.

Comparative toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in four (sub)strains of adult male rats.

Four (sub)strains of adult male rats were given single oral doses of various concentrations of TCDD to establish and compare the oral 30-day LD50 values. The strains of rats were Fischer (F/334N) supplied by Harlan Industries, Frederick Cancer Research Center, and Charles River Breeding Laboratories; and CD supplied by Charles River Breeding Laboratories. The Charles River/Fischer rats were most sensitive to TCDD (LD50 = 164, 95% confidence limits 104-217 micrograms/kg), the Frederick/Fischer and Charles River/CD rats were moderately sensitive to TCDD (LD50 = 303, 250-360; and 297, 240-360 micrograms/kg, respectively), and the Harlan/Fischer rats were most resistant to TCDD (LD50 = 340, 281-409 micrograms/kg). The mean times of death were from 24.5 +/- 1.0 to 28.3 +/- 0.5 days and the percentage body weight loss at death was 37.4 +/- 1.2 to 42.7 +/- 1.3%. One week after exposure of the Charles River/Fischer animal to 45 micrograms TCDD/kg (1/4 the established 30-day LD50 dose), the same serum profile was induced as previously observed in the Harlan/Fischer rat, which includes hypoglycemia, hypertriglyceridemia, and hypercholesterolemia. These results emphasize the importance of indicating the precise dose, strain of rat, and time after dosing before termination in reporting the effects of TCDD on a particular biological response.

Dose-related effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57BL/6J and DBA/2J mice.

The dose-related effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied in B6D2F1/J (B6D), C57BL/6J (C57), and DBA/2J (DBA) mice. A 14-fold difference in lethality was observed in C57 and DBA mice, based upon 30-day LD50 values of 182 and 2570 micrograms TCDD/kg body wt, respectively. The 30-day LD50 for B6D mice was 296 micrograms TCDD/kg body wt. A progressive loss of body weight in all strains of mice was observed during the 30-day LD50 studies, with maximal weight losses of 24.7, 34.0, and 33.4% prior to death of C57, B6D, and DBA mice, respectively. In separate experiments, it was found that decreased feed consumption did not contribute to weight loss in C57 mice exposed to lethal or sublethal doses of TCDD until the animals were moribund. Time-course studies in C57 mice treated with 200 micrograms TCDD/kg body wt indicated that decreases in serum glucose and triglyceride concentrations and increases in hepatic triglyceride content occurred within 4 to 8 days of exposure, and were maximally altered within 17 to 21 days postexposure, concomitant with a 25% body weight loss. C57 mice fasted for 24 to 96 hr lost 18% of body weight and also exhibited alterations in glucose and lipid parameters; however, these changes were substantially different than the effects of TCDD exposure. In concert, these observations demonstrate that decreased feed consumption (hypophagia) does not account for weight loss and changes in carbohydrate and lipid metabolism in TCDD-treated C57 mice. Dose-response experiments resulted in comparable changes in glucose and lipid parameters when DBA mice were exposed to 10-fold higher doses of TCDD than C57 mice. Parallel LD50 responses and parallel changes in carbohydrate and lipid metabolism, at 10- to 15-fold differences in dose range, are indicative of a common mechanism of toxicity in TCDD-treated C57 and DBA mice.

Effect of phthalate esters on energy coupling and succinate oxidation in rat liver mitochondria.

Isolated rat liver mitochondria were exposed to mono- and di-n-butyl phthalate (MBP and DBP) and mono- and di(2-ethylhexyl)phthalate (MEHP and DEHP) and examined for effects on mitochondrial energy-dependent processes, including oxidative phosphorylation and active K+ uptake. Additional studies on the effects of these phthalate esters on succinate oxidation and on mitochondrial membrane integrity are also included. DBP and MEHP stimulated succinate state 4 respiration, impaired K+-valinomycin induced swelling with succinate, ascorbate, or ATP as the energy sources, and inhibited succinate state 3 respiration and succinate cytochrome c reductase activity. MEHP was found to act as a non-competitive inhibitor of succinate dehydrogenase activity, with an apparent Ki = 2.4 X 10(-4) M. At concentrations which uncouple energy linked reactions, MEHP and DBP produced only slight energy-independent swelling and release of soluble proteins from isolated mitochondria. MBP caused only slight stimulation of state 4 respiration and impairment of K+-valinomycin induced swelling with each of the 3 energy sources, however, of the 4 phthalate esters, it produced the greatest energy-independent swelling and led to the greatest release of soluble mitochondrial proteins. DEHP had no apparent effect on any of these processes except for slight impairment of ATP-dependent K+-valinomycin induced swelling. It is concluded that phthalate ester toxicity in liver mitochondria is due to uncoupling of energy linked reactions and/or inhibition of succinate dehydrogenase activity. Uncoupling by MBP may involve disruption of mitochondrial membrane integrity, while uncoupling by DBP and MEHP is probably due to an increase in membrane permeability to H+ and other small ions.

Development of rat liver and colon substrate cycle enzymes in offspring with and without prenatal exposure to a carcinogenic dose of 1,2-dimethylhydrazine.

Pregnant F-344 rats were exposed by intubation to a single dose (35 mg/kg) of 1,2-dimethylhydrazine dihydrochloride or to an acetate buffer on day 14 of gestation. A detailed examination of the effects of this dose of 1,2-dimethylhydrazine on brush border enzymes in the offspring was performed. Changes in the liver and colon levels of the alpha-glycerophosphate and malate/aspartate substrate cycle enzymes were measured during the development; at days 17 and 20 of gestation and at 2, 6, 13, 20, 27, 55, 110 days and 1 year after birth. It is concluded that metabolic energy enzymes, glutamate oxaloacetate transaminase and malate dehydrogenase, are more sensitive to 1,2-dimethylhydrazine treatment than are the brush border hydrolases or NAD- and Fp-linked alpha-glycerophosphate dehydrogenases.

Mitochondrial toxicity of phthalate esters.

The effects of mono- and dibutyl phthalate and mono- and di(2-ethylhexyl) phthalate on energy-dependent K+ uptake, respiration rates, and succinate cytochrome c reductase activities of isolated rat liver mitochondria were evaluated. The energy-coupling processes, active K+ transport and oxidative phosphorylation, were affected most by di-n-butyl phthalate and mono(2-ethylhexyl) phthalate. Mono-n-butyl phthalate had a moderate effect on energy coupling and di(2-ethylhexyl) phthalate had no apparent effect. The potency of inhibition of succinate cytochrome c reductase activity was mono(2-ethylhexyl) phthalate greater than di-n-butyl phthalate greater than mono-n-butyl phthalate = di(2-ethylhexyl) phthalate. It is concluded that phthalate esters affect mitochondrial activities by altering the permeability properties of the inner membrane and by inhibiting succinate dehydrogenase activity.

Interaction of aromatic aldehydes with isolated rat liver mitochondria.

It has been suggested that aromatic aldehydes may reduce cytochrome c [Wolf et al. Fedn Proc. 39 (3), 1013 (1980)]. Therefore, interaction of the aromatic aldehydes, p-anisaldehyde, benzaldehyde, p-tolualdehyde, p-carboxybenzaldehyde, p-chlorobenzaldehyde and p-nitrobenzaldehyde, with rat liver mitochondria was examined in vitro. Although both pyruvate/malate- and succinate-mediated respiration, as well as that mediated by other citric acid cycle intermediates, were inhibited by the aromatic aldehydes (0.5 to 1.0 mM), cytochrome c oxidase was not inhibited by aromatic aldehydes (1.0 to 20 mM). There was a marked inhibition of succinic dehydrogenase and both ADP- and DNP-stimulated respiration by benzaldehyde (2 to 20 mM). Since both pyruvate/malate- and succinate-mediated respiration were inhibited by the aromatic aldehydes without inhibition of cytochrome c oxidase, several sites of inhibition, possibly both at the site of transport of substrates and the active enzymes, may exist. Benzaldehyde, 300 microM, inhibited pyruvate/malate-mediated state 3 respiration by 50% which suggests that no additional functional group or metabolism to another species is required for these inhibitory effects.

Effects of polychlorinated biphenyls on the development of intestinal and serum marker enzymes.

The effects of polychlorinated biphenyls (PCBs) on the development of several intestinal and serum marker enzymes have been studied. The three congeners 4-monochloro (1-CB), 3, 4, 3',4'-tetrachloro (4-CB), and 2, 4, 5, 2', 4, 5'-hexachloro (6-CB) biphenyl were administered orally to pregnant rats on d 8, 11, 13, 15, and 18 or gestation. 1-CB and 6-CB were intubated at doses of 30 mg/kg.d (total dose, 150, mg/kg) and 4-CB was administered at 3 mg/kg-d (total dose, 15 mg/kg). Levels of intestinal alkaline phosphatase, monoamine oxidase, and Na+, K+-adenosin-5'-etriphosphatase and levels of serum alkaline phosphatase, sorbitol dehydrogenase, and beta-hydroxybutyrate dehydrogenase were measured in the dams after weaning and in their offspring at -1, 6, 20, and 55 d of age. Intestinal alkaline phosphatase activity was elevated at the later postnatal stages in the 1-CB group and depressed at 55 d in the 4-CB group, whereas serum alkaline phosphatase levels were markedly depressed prenatally and postnatally in the 4-CB and 6-CB groups, respectively, Intestinal monoamine oxidase levels were markedly increased in the 6-CB group at -1, 6, and 20 d of age and significantly depressed in the 4-CB animals at -1 and 55 d of age. There was an increase in monoamine oxidase activity in the 4-CB group at 6 d. The 1-CB group exhibited depression of monoamine oxidase levels at 6 and elevation at 20 and 55 d. Intestinal Na+, K+-ATPase levels were elevated throughout development in the 1-CB animals and at -1 and 6 d in the 4-CB group. The 6-CB animals showed elevated levels of Na+, K+-ATPase only at 6 d. Serum beta-hydroxybutyrate dehydrogenase and sorbitol dehydrogenase were induced prenatally in the 4-CB animals but enzyme activities decreased to normal by 55 d of age. Significant depression of activity was evident in the 1-CB and 6-CB groups at -1 d in both enzymes. Dams in the 1-CB group showed significant changes in intestinal monoamine oxidase, serum sorbitol dehydrogenase, and serum alkaline phosphatase. Serum levels were elevated in the 4-CB group. Activities of intestinal enzymes remained unchanged in the 4-CB group. All maternal enzyme levels monitored were not significantly changed in the 6-CB group.

Utilization of blood analyses to evaluate metabolic changes in control and 1,2-dimethylhydrazine-treated adult male fischer rats.

The synthetic compound 1,2-dimethylhydrazine is employed in carcinogenesis studies because of its reliable and specific ability to produce colon tumors in rodents. Male Fischer rats were treated at 7 weeks of age with a single oral dose of 1,2-dimethylhydrazine (35 mg/kg) and examined at autopsy 1.5 years later when the incidence of colon tumors is approximately 80%. Blood from control and 1,2-dimethylhydrazine-treated animals was taken at autopsy for routine hematoplazia analysis and for biochemical analysis with the Sequential Multiple Analyzer Computer multitest system. The results indicate that the induction of tumors with a single oral dose of this carcinogen is associated with statistically significant changes in the serum levels of some clinically useful metabolic parameters. Clinically significant changes in the serum chemistry were increases in the creatine phosphokinase (CPK) and albumin/globulin values without an increase in the total serum protein. The multitest system has not been previously employed to evaluate the blood chemistry profiles of tumor-bearing animals and, thus, this study provides an illustration of the potential for this technique to evaluate metabolic changes associated with exposure to carcinogens.

Induction of colon tumors by a single oral dose of 1,2-dimethylhydrazine.

Male Fischer rats were treated at 7 weeks of age with a single oral dose of 1,2-dimethylhydrazine (35 mg/kg). After 1.5 years, the 14 control and 28 treated animals were killed for general autopsy. The incidence of tumor formation in the treated animals was 78.6% as compared to 0% for the control animals. All tumors (1--3/rat) were located in the colon, with the exception of one in the Zymbal's gland of the ear and one in the small intestine. The dosage of 1,2-dimethylhydrazine used in this study is the lowest single oral dose of this carcinogen reported to induce colon tumors.