[Show abstract][Hide abstract] ABSTRACT: In continuing studies of limb effects resulting from fetal exposure to N(G)-nitro-(L)-arginine methyl ester (L-NAME), we examined the early time course of vascular changes and the effectiveness of fetal intraamniotic injection. Vascular engorgement and hemorrhage occurred within 4 hr of L-NAME treatment on gestational day (gd) 17, and direct injection appeared to be as effective as maternal intraperitoneal injection in inducing limb hemorrhage. Further studies examined protein nitration and electron transport inhibition in tissues of exposed fetuses. L-NAME caused significant increases in nitrotyrosine (NT) formation in limb but not in heart or brain, and reduced electron transport rates in limb. Three agents, alpha-phenyl-N-t-butylnitrone (PBN), a radical trap and inhibitor of inducible nitric oxide synthase (iNOS), allopurinol, an inhibitor of xanthine oxidase, and aminoguanidine, a relatively specific inhibitor of iNOS, significantly moderated limb hemorrhage and protein nitration in distal limb. These results suggest that L-NAME works directly on the fetal limb vasculature and indicate a cytotoxic role for peroxynitrite, a potent oxidant and nitrating agent that is the reaction product of nitric oxide and superoxide anion radical. We propose that L-NAME and other vasoactive toxicants disrupt the fetal limb in a sequential process. Initially, nitric oxide (NO) is depleted, causing hemorrhage and edema in the limb. Within hours, iNOS is induced, resulting in cytotoxic tissue concentrations of NO and reactive nitrogen species that induce apoptosis and/or necrosis in the limb. We suggest that L-NAME exposure may serve as a model of vascular disruptive limb malformations.
[Show abstract][Hide abstract] ABSTRACT: It is well recognized that reactive oxygen species (ROS) are formed during the reperfusion of ischemic tissues and ROS may be pathogenic in adult tissues. Although there is little information on the formation and toxicity of ROS during prenatal life, a strong association has been made between limb and possibly brain malformations and uteroplacental ischemia during fetal stages of gestation. It has been proposed that these malformations result from attack by ROS formed during the resumption of placental perfusion. Studies reported here examined formation of ROS in teratogenically sensitive limb and brain and insensitive heart before and during the period of teratogenic sensitivity. Also examined was the formation of ROS following hypoxia and reoxygenation in fetal culture and DNA hydroxylation in sensitive and insensitive fetal tissues during uteroplacental ischemia and reperfusion in vivo. Rates of formation of superoxide anion radical and hydrogen peroxide declined with increasing gestational age whereas those for hydroxyl radical increased. Hydrogen peroxide generation was greatest in insensitive heart whereas hydroxyl radical formation was significantly lower in brain than in limb or heart, which had comparable rates. Hydrogen peroxide generation, which declined significantly in fetuses, but not in membranes with gestation, failed to respond to reoxygenation in vitro. Finally, there were significant increases in DNA hydroxylation in fetal hearts and limbs, but not in brains during uteroplacental ischemia but no further significant change could be detected after reperfusion.
No preview · Article · Aug 1998 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: Activities of three types of superoxide dismutase in tissue fractions were significantly lower in fetal and adult brain and fetal limb preparations than in fetal and adult heart preparations. An exception was the cytoplasmic fraction of adult brain that had levels of Cu, Zn-superoxide dismutase activity comparable to those in cytoplasmic fractions of heart. In addition, Mn superoxide dismutase activity appeared to be very low in all fetal mitochondrial matrix fractions and cytoplasmic fractions as well as in adult brain. Finally, the results of these studies emphasize the importance of two antioxidant defense systems in the tissues studied, one associated with the mitochondrial electron transport system and the other, the cytosolic Cu, Zn enzyme.
No preview · Article · Mar 1998 · Free Radical Research
[Show abstract][Hide abstract] ABSTRACT: While the limb bud and brain of the rat develop abnormally in response to transient uteroplacental hypoperfusion during late gestation, the heart appears to be protected. These malformations have been associated with the generation of reactive oxygen species (ROS). Studies were designed to examine superoxide generation by mitochondrial electron transport particles (ETP) from adult and conceptal tissues and to investigate characteristics that could be responsible for heightened concentrations of ROS in sensitive tissues. Parameters investigated included NADH oxidase and cytochrome c oxidase activities, cytochrome content, and superoxide dismutase activity.
NADH oxidase activities were significantly lower in sensitive tissues that also developed the highest concentrations of superoxide. Because ETP from adult CNS also had low NADH oxidase activity but did not show increased concentrations of superoxide, inhibition of electron transport did not adequately account for increased ROS concentrations. The reduced NADH oxidase activity of sensitive tissues could not be caused by inhibition at the cytochrome c oxidase region since this latter activity equaled or exceeded the former in all instances. No significant differences were found in the cytochrome contents of different tissues. There was significantly less superoxide dismutase activity in homogenates prepared from either of the two sensitive conceptal tissues compared with those from insensitive conceptal or adult tissues.
These studies confirm the presence of heightened concentrations of superoxide anion radical in ETP from teratogenically sensitive tissues and suggest that these concentrations may result primarily from decreased activity of superoxide dismutase(s) in those tissues. Superoxide anion radical could therefore be available to participate in the generation of the more toxic oxidant species such as the hydroxyl radical.
[Show abstract][Hide abstract] ABSTRACT: It has been shown that multiple exposures of gravid rats to cocaine during late gestation result in significant incidences of severe malformations. Hind limb reduction defects were frequent findings in this study. Other studies have shown that comparable abnormalities can be induced in experimental animals by various procedures including vascular clamping, direct fetal exposure to epinephrine, uterine handling following laparotomy, as well as by exposure to hyperbaric oxygen. This paper reviews these and other studies, and presents a novel mechanistic hypothesis that explains their common findings. It is proposed that in each instance, conceptual hypoxia results from hypoperfusion caused by transient vasoconstriction. Following the resumption of normal perfusion, reactive oxygen species are generated by the ischemia/reperfusion mechanisms thought to underlie many pathobiologic lesions. It is proposed that the conceptus is particularly vulnerable to the toxicity of oxygen radicals because of its low antioxidant activities and the highly reduced state of its undifferentiated cells. Sensitivity to cocaine and uterine handling appears to be enhanced during late gestation and it is hypothesized that this results from changes in oxygenation and iron content that increase both the substrate and catalyst for generation of reactive oxygen species.
[Show abstract][Hide abstract] ABSTRACT: The administration of multiple doses of cocaine on a single day during late gestation is teratogenic in rats in which hind limb ectrodactyly is a major finding (Webster and Brown-Woodman, '90). We have previously hypothesized that these limb malformations result from the generation of reactive oxygen species during the process of ischemia/reperfusion in vivo. In order to study the direct effects of cocaine versus the aberrant oxygenation it may induce, we have developed a system for culturing rat embryos between days 14 and 15 of gestation. Growth and development of cultured embryos are comparable to that of in vivo controls. Exposure to normoxia (95% O2) with or without cocaine failed to induce limb malformations and exposure to a single long period of hypoxia (20% O2) only reduced limb growth in the anterior-posterior axis. By contrast, embryos receiving multiple brief exposures to hypoxia developed a significant incidence of hind limb ectrodactyly that appeared indistinguishable from that induced by cocaine in vivo. By incubating day 14 embryos in a nitroblue tetrazolium derivative, 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), it was shown that superoxide anion radical appears in the digital rays following two episodes of reperfusion. Little reaction product was seen under the other conditions. Finally, mitochondrial electron transport particles prepared from teratogenically sensitive limb buds spontaneously "leak" electrons to form superoxide anion radical whereas those from insensitive heart fail to do so. We propose that cocaine and other exposures that can transiently reduce conceptual oxygenation during late gestation are teratogenic by virtue of their capacity to induce ischemia/reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)
[Show abstract][Hide abstract] ABSTRACT: Previous study has shown that midorganogenesis-stage rat embryos exposed to strong redox cyclers under moderate hypoxia in vitro develop severe necrotic defects on the right side. Similar effects can be produced by exposure to severe hypoxia alone. Studies presented here indicate that exposure to severe but survivable hyperoxia induces comparable necrotic degeneration on the left sides of all embryos. We hypothesize that the basis of these axially asymmetric defects is relatively precocious mitochondrial maturity on the left side of the embryo. In order to investigate this hypothesis, we compared mitochondrial oxygen utilization (NADH oxidase activities) on either side of rat embryos between days 11 and 14 of gestation. Activities were consistently higher on the left side during this period and significantly higher on day 11. We also found that the asymmetric embryotoxicity induced by niridazole, a strong redox cycler, could be attenuated by prior culture under hyperoxic conditions. We propose that mitochondrial immaturity on the right results in inadequate energy generation under hypoxic conditions, either directly or as a result of redox cycling. On the other hand, necrosis associated with hyperoxic conditions results from "leakage" of superoxide from functionally mature mitochondria on the left side.
[Show abstract][Hide abstract] ABSTRACT: Day 10 rat embryos were exposed to cocaine HCl (10–100 μM) in vitro in 20% (designated normoxic) and 10–12% (designated moderately hypoxic) oxygen and examined the following day. In normoxia, it caused prompt and significant decreases in heart rates and significant reductions in measures of growth and development and diameters of the vitelline arteries. In moderate hypoxia, cocaine exposure resulted in axially asymmetric defects reported previously only in embryos exposed to extreme hypoxia or to hypoxia generated by redox cyclers.
Day 10 or 11 embryos or isolated hearts from the latter stage were incubated with cocaine under normoxic conditions. Acute and significant concentration-dependent decreases in heart rates occurred on day 10. The rates in day 11 embryos and in isolated hearts from day 11 embryos were less sensitive than those on day 10. Cocaine also significantly inhibited the activity of the terminal electron transport system of the mitochondria of embryos.
Maternal cocaine exposure has been associated with uterine vasoconstriction and decreases in fetal oxygenation. The latter has been shown to stimulate glucose uptake. We hypothesize that placental vasoconstriction limits the ability of embryos to meet the increased glucose demands induced by hypoxia. The developmental toxicity of nutrient and oxygen deprivation is further enhanced by significant decreases of mitochondrial activity. We propose therefore that compromised energy supplies form the basis of the developmental toxicity of cocaine.
[Show abstract][Hide abstract] ABSTRACT: A summary is presented of previous studies by other investigators of human achondroplasia and dyschondroplastic animal models. In addition, studies previously reported from our laboratories are discussed, and they demonstrate that defective oxidative energy metabolism is present in mitochondrial preparations from achondroplastic human subjects and rabbits (ac/ac) with chondrodystrophy. The results of the studies support the hypothesis discussed fully in the manuscript that a partial defect in mitochondrial oxidative metabolism in achondroplastic subjects is expressed specifically in the growth plates of the long bones because this tissue has the lowest oxygen tension of any bodily organ undergoing active proliferation, thus leading to the achondroplastic phenotype in humans and the ac/ac rabbit. In the ac/ac rabbit phosphorylation at the cytochrome c oxidase region (site III) of the terminal respiratory system was shown to be absent in mitochondrial preparations from the livers of newborn ac/ac rabbits. Normal-appearing littermates did not exhibit the defect. Studies of mitochondrial preparations from human skin fibroblasts (grown in tissue culture) from normal human subjects and subjects with homozygous achondroplasia demonstrated that concentrations of cytochrome a3 were decreased approximately 80% in preparations from homozygous achondroplastic cells. Levels of cytochrome a3 in heterozygous achondroplastic cells were intermediate between the levels in normal cells and homozygous achondroplastic cells demonstrating the effects of gene dosage. Determination of total heme a (as the pyridine hemochromogen) in the normal and achondroplastic preparations from human subjects showed that the observed decrease in concentration of cytochrome a3 in the achondroplastic preparations was due to an absence of cytochrome a3 and not to a change in its absorbancy (extinction coefficient).
[Show abstract][Hide abstract] ABSTRACT: Mitochondria prepared from tissue culture cells (skin fibroblasts) from normal subjects and subjects with homozygous achondroplasia were studied to determine the concentrations of cytochromes a and a3 in the preparations. Cytochrome a3 was markedly decreased (80%) in the achondroplastic preparations with cytochrome a present in normal amounts. Determination of total heme a (as the pyridine hemochromogen) in the normal and achondroplastic preparations demonstrated that the observed decrease in concentration of cytochrome a3 in the achondroplastic preparations was due to an absence of cytochrome a3 and not to a change in its absorbancy (extinction coefficient). The decreased concentrations of cytochrome a3 in the achondroplastic cells may decrease the reactivity or affinity of the mitochondrial oxidative systems for oxygen and result in the phenotypic expression of the disease.
No preview · Article · May 1987 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: Mitochondria prepared from the first growth of cells (fibroblasts) from skin biopsies from homozygous (but not heterozygous) achondroplastic human subjects were unable to carry out oxidative phosphorylation. However, successive crops of cells gained the ability to phosphorylate with normal P:O ratios with pyruvate-malate and succinate as substrates. Concentrations of cytochromes a + a3 were markedly and significantly lower in homogenates of homozygous achondroplastic tissue culture cells than in homogenates of normal cells. Levels of cytochromes a + a3 in the heterozygous achondroplastic cells were intermediate between the levels in normal cells and the homozygous achondroplastic cells. Activities of the mitochondrial oxidative systems (NADH, succinic and cytochrome oxidases) were not significantly lower in the achondroplastic cell preparations than in normal cell preparations under standard assay conditions (saturation levels of oxygen).
[Show abstract][Hide abstract] ABSTRACT: Though previously described as very low or absent in yeast, we find significant pyridine nucleotide transhydrogenation (NADPH + acetyl pyridine-NAD+----NADP+ + acetyl pyridine-NADH) activity in yeast extracts when assayed at pH 8-9, and describe here the subcellular distribution and separation of the various molecular forms contributing to the total activity in two yeast species. Gentle subcellular fractionation reveals transhydrogenase activity only in the cytosolic fraction of both Saccharomyces cerevisiae and Candida utilis while intact mitochondria and microsomes are without activity. On sucrose gradient centrifugation, this soluble cytosolic activity proves to be primarily in a high-molecular-weight (greater than 10(6)) band which has salmon-colored fluorescence on uv illumination. Sonication of the particulate subcellular fractions solubilizes substantial transhydrogenase activity from mitochondria of C. utilis (but not from S. cerevisiae) which on sucrose gradients consists of both high (greater than 10(6))- and low-molecular-weight active fractions, each with yellow-green fluorescence. Ammonium sulfate fractionation and sucrose gradient centrifugation of protein solubilized from whole yeast of both species by vigorous homogenization with glass beads confirms the presence and fluorescence of these various molecular weight forms. The relationship of these activities to other enzymatic activities (especially the mitochondrial external NADH dehydrogenase) is discussed.
No preview · Article · Jan 1986 · Archives of Biochemistry and Biophysics
[Show abstract][Hide abstract] ABSTRACT: Mitochondria were prepared from the brown adipose tissue of control rats and animals made iron deficient by means of a low iron diet. The specific activities of the mitochondrial electron transport system (NADH, succinate and alpha-glycerophosphate oxidase activities) were markedly and significantly reduced in preparations of brown adipose tissue from the iron-deficient rats as compared with preparations from the control animals. In contrast, concentrations of the cytochrome pigments a + a3, and c + c1 were normal and cytochrome b was slightly reduced (18%) in the mitochondrial preparations from the iron-deficient animals. Treatment of the iron-deficient animals with triiodothyronine significantly increased the amount of brown fat present per kilogram of body weight in both control and iron-deficient rats, but did not significantly affect the specific activities of the mitochondrial electron transport system.
[Show abstract][Hide abstract] ABSTRACT: An NADH cytochrome c reductase has been identified in plasma membrane fractions from neutrophils in addition to the superoxide producing NADPH oxidase which has been extensively studied by other investigators. Activation of neutrophils resulted in increased enzyme activities but to different degrees; the NADH cytochrome c reductase increased 2 fold in specific activity and the NADPH oxidase 30 fold. Treatment of the plasma membrane fraction with sonication and differential centrifugation yielded a particulate fraction (R2) with a 2 fold increase in specific activities of both enzymes and concentrations of cytochrome b and FAD. The cytochrome b in the preparation was not reduced under anaerobic conditions by either NADH or NADPH. Treatment of preparations of R2 with deoxycholate or potassium thiocyanate separated the two enzymes yielding particulate preparations with only NADPH oxidase or NADH cytochrome c reductase activity, respectively.
No preview · Article · Oct 1985 · Biochemistry international
[Show abstract][Hide abstract] ABSTRACT: Submitochondrial particles prepared from liver and skeletal muscle of control and iron-deficient rats were examined for cytochrome content and for both energy-independent and energy-conserving functions. Liver submitochondrial particles appear quite resistant to iron deficiency with cytochrome content and electron-transferring or energy-conserving functions maintained at a level of 85% or better of normal. Iron-deficient skeletal muscle submitochondrial particles, in contrast, have decreased cytochrome content and only 15-20% of the normal capacity for oxidation through either complex I (NADH dehydrogenase) or complex II (succinate dehydrogenase). Energy-linked reactions which involve substrate oxidation/reduction (succinate----NAD+ reversed electron flow and succinate-driven energy-dependent transhydrogenation) are likewise markedly decreased, while ATP-driven energy-dependent transhydrogenation and mitochondrial ATPase are normal. Our data support the concept that iron deficiency leads to decreased electron-carrying capacity of iron-containing mitochondrial enzymes, with skeletal muscle being much more susceptible than liver, but that the mitochondria are otherwise normal with regard to energy conservation.
No preview · Article · Sep 1985 · Biochemical Medicine
[Show abstract][Hide abstract] ABSTRACT: Gravid Sprague-Dawley-derived rats were injected SC twice daily with either 20 or 40 mg/kg pentobarbital sodium (PT), sodium phenobarbital (PH), or the same volume of the saline vehicle on days 9-21 of pregnancy. Pair-feeding was employed. Vital, developmental, and activity measures were obtained on the neonates and locomotor activity was measured from 3-10 months of age. Avoidance was measured sequentially in a shuttle box, and in an operant chamber beginning at 3 months of age. The PH-80 dams gained less weight over the gestational period, and PH-80 and PH-40 offspring had more neonatal deaths. These male offspring were hyperactive at maturity, and PH-80 rats were initially slower to escape experimenter-initiated shock. PT exposure caused transient neonatal and juvenile hyperactivity. PT rats performed more poorly on both the conditioned avoidance and Sidman shock schedules, and had significantly lower brain: body weight ratios at 1 year of age. All four drug groups outperformed the saline offspring on subject-initiated shock schedules (punishment). Sex of offspring was determined on postnatal day 4 and the sex ratio was shifted towards male births with both drugs relative to controls.
No preview · Article · Feb 1985 · Psychopharmacology
[Show abstract][Hide abstract] ABSTRACT: Studies were performed to determine the effects of iron deficiency in the rat on neutrophil activation and on levels of neutrophil myeloperoxidase and cytochrome b. The period of time required for neutrophil activation was not significantly affected by iron deficiency, but the maximum rates of respiration attained after activation were markedly lower (60% decrease) in iron-deficient neutrophils than in control cells. The myeloperoxidase activity of neutrophils from iron-deficient rats was also markedly decreased (approximately 75%) compared with the activity of control cells; however, the concentration of cytochrome b in the neutrophils was unaffected by iron deficiency.