Zinc,ethanol, and lipid peroxidation in adult and fetal rats

Studies were performed on adult and fetal rats receiving either a zinc-deficient (<0.5 ppm) diet and/or ethanol (20%) throughout pregnancy. Liver zinc levels were depressed in fetuses exposed toin utero zinc deficiency, but brain zinc levels were unchanged. Ethanol had no effect on the concentration of zinc in the several fetal and adult tissues studies. Lipid peroxidation, as measured by endogenous levels of malondialdehyde (MDA) increased following food restriction, zinc improverishment, and alcoholism in adult and fetal livers, but not in fetal brains. Generally, levels of MDA were highest when both zinc deficiency and the ingestion of alcohol occurred concurrently. Glutathione (GSH) was depressed by zinc restriction in several adult and fetal tissues, but not in the fetal brain. Ethanol alone had no effect on GSH levels. The activity of the enzyme glutathione peroxidase (GSH-Px) was not changed in either organism by alcohol or zinc deficiency. Overall, the data point to increased lipid peroxidation in maternal and fetal rat tissues following zinc depletion and/or treatment with alcohol and draw attention to the apparent vulnerability of the fetal liver toin utero alcoholism. By contrast, the fetal brain seems to be especially resistant to alcohol and zinc-related lipoperoxidation. An association is suggested between the increased lipoperoxidation accompanying zinc deficiency and reduced levels of GSH, but this does not appear to relate to changes in the activity of GSH-Px. A similar relationship is not evident with respect to the increased levels of MDA in fetal and adult livers following chronic alcohol intoxication. A possible basis for the zinc-GSH interaction is discussed.     

Hexokinase isoenzymes in tissues of the adult and developing guinea pig

Changes in the activities and isoenzyme distribution of hexokinase were determined in a number of tissues during the development of the guinea pig. The total activity in the fetal liver showed a large fall during the second half of gestation to reach adult values by term. With normal diet the fetal, neonatal, and adult livers had isoenzymes I and III but little or no detectable IV (glucokinase). The fetal liver had predominantly type I, but the proportion of type III increased during development. The kinetics of the guinea pig isoenzymes were similar to those reported for the rat. Two additional isoenzymes with mobility between I and II were detected in the fetal liver and blood. They appear to have kinetic properties similar to type I. Detectable liver glucokinase activity was induced by glucose administration to adult guinea pigs. The total activity in kidney, brain and skeletal muscle showed a postnatal rise while in the fetal heart it was high and declined after birth. These tissues contained predominantly type I with varying proportions of type III hexokinase. The ratio of particulate-bound to soluble hexokinase varied from tissue to tissue. All except the liver showed a significant increase in binding after birth. The changes are discussed in relation to the control of glucose utilization in the fetal and neonatal periods.     

Adenylate kinase isoenzyme patterns in normal and neoplastic human lung, and in various adult as compared to fetal rat tissues

The isozyme pattern and total activity of adenylate kinase were studied in normal adult and fetal human and rat tissues using starch gel electrophoresis. Three adenylate kinase isoenzymes were identified in human tissues. Although normal adult lung exhibited higher adenylate kinase activity than did its fetal or neoplastic variant, isozyme patterns in the three types of tissues were indistinguishable from each other and from that in fetal human liver. The pattern of these three isozymes in rat lung (as in spleen) also did not change between fetal and adult life. However, adult kidney and heart of this species did appear to contain isozymes not present in fetal life. Brain (both adult and fetal) was striking different from all the other tissues in that it contained only one adenylate kinase isozyme. The total adenylate kinase activity per gram of adult rat liver, kidney and lung was significantly higher than in the cognate fetal organs, whereas that in brain or spleen did not change with age. The activity in adult heart (similar to the fetal one) was higher than in any other tissue examined.     

Patterns of alternative splicing of fibronectin pre-mRNA in human adult and fetal tissues

Alternative splicing of fibronectin pre-mRNA at two distinct regions, termed ED-A and IIICS, was investigated with human adult and fetal tissues by the nuclease S1 protection assay. A clear tissue specificity was observed in the splicing pattern at the ED-A region. More ED-A+ than ED-A- mRNAs were identified in lung, whereas ED-A- mRNAs were predominantly expressed in liver. Endometrium contained nearly equal amounts of ED-A+ and ED-A- mRNAs. The splicing pattern at the ED-A region was also different between adult and fetal liver but not between adult and fetal lung. Tissue type specific splicing was also observed at the IIICS region. Although the mRNA species containing the complete IIICS sequence comprised 40-65% of the total fibronectin mRNAs irrespective of tissue types, expression of the mRNA species lacking a part or all of the IIICS sequence was more pronounced in adult liver than in other tissues including fetal liver. These results strongly suggest that the alternative splicing of fibronectin pre-mRNA in vivo is regulated in a tissue type specific manner at both the ED-A and IIICS regions and that it is developmentally regulated in liver but not in lung. On the basis of these and other observations reported previously, a possibility that a part of the fibronectins synthesized and secreted by hepatocytes is deposited in the tissue matrix is discussed.     

The tissue-specific expression and developmental regulation of two nuclear genes encoding rat mitochondrial proteins. Medium chain acyl-CoA dehydrogenase and mitochondrial malate dehydrogenase

To study the regulation of nuclear genes which encode mitochondrial enzymes involved in oxidative metabolism, absolute levels of mRNA encoding rat medium chain acyl-CoA dehydrogenase (MCAD) and rat mitochondrial malate dehydrogenase (mMDH) were determined in developing and adult male rat tissues. MCAD mRNA is expressed in a variety of adult male tissues with highest steady state levels in heart, adrenal, and skeletal muscle and lowest levels in brain, lung, and testes. In comparison, steady state levels of mMDH mRNA in adult male rat tissues were similar to those of MCAD mRNA in heart, small intestine, adrenal, and skeletal muscle but markedly different in brain, stomach, and testes. Thus, the steady-state levels of MCAD and mMDH mRNA are highest in adult tissues with high energy requirements. Dot blot analysis of RNA prepared from late fetal, suckling, and weaning rat heart, liver, and brain demonstrated the presence of MCAD and mMDH mRNA during the fetal period in all three tissues. Both MCAD and mMDH mRNA levels increased 2-2.5-fold at birth followed by a decline during the first postnatal week in heart and liver. The patterns of accumulation of these mRNAs in heart and liver during the weaning and early adult periods were also similar, although the absolute levels were significantly different. Brain MCAD mRNA levels were consistently low (less than 0.1 pg/micrograms total cellular RNA) throughout the developmental stages. However, brain mMDH mRNA levels exhibited a marked increase during the weaning period, reaching a peak concentration which is higher than the level of mMDH mRNA in heart and liver at any point during development. These results indicate that the level of expression of the nuclear genes encoding MCAD and mMDH is tissue-specific and developmentally regulated. The patterns of MCAD and mMDH mRNA accumulation parallel the changes in energy metabolism which occur during development and among adult tissues.     

Parathion and methyl parathion toxicity and metabolism in piperonyl butoxide and diethyl maleate pretreated mice

Pretreatment of male mice with piperonyl butoxide, 400 mg/kg 1 h before challenge with insecticides, resulted in a 40-fold antagonism of the acute i.p. toxicity of methyl parathion but potentiated the toxicity of parathion two-fold. Piperonyl butoxide had no effect on the toxicity of the oxygen analogs of these insecticides, methyl paraoxon and paraoxon. Diethyl maleate (1 ml/kg) depleted liver glutathione by 80% after one hour, potentiated the toxicity of both methyl parathion and methyl paraoxon, and partially counteracted the protective effect of piperonyl butoxide on methyl parathion toxicity. Piperonyl butoxide delayed the onset of brain cholinesterase inhibition by parathion. Studies of the metabolism of the insecticides by liver homogenates in vitro demonstrated that piperonyl butoxide inhibited both the oxidative formation of the oxygen analogs (activation) and oxidative cleavage to p-nitrophenol and dialkylphosphorothioic acid (detoxification). While parathion metabolism was mostly oxidative, methyl parathion metabolism appeared to be predominantly via glutathione-dependent enzymes. Studies of in vitro distribution of the insecticides demonstrated that piperonyl butoxide pretreatment resulted in elevated tissue concentrations of parathion and methyl parathion; however, the rate constant for elimination from plasma for both insecticides was unaffected by piperonyl butoxide. The overall rate of metabolism of methyl parathion in vivo was approximately twice that of parathion. These results suggest that during piperonyl butoxide inhibition of oxidative activation and cleavage, methyl parathion detoxification continues through uninhibited glutathione-dependent pathways of metabolism. The net result is a reduction in the acute toxicity of methyl parathion. Lack of an effective alternate pathway of detoxification may explain the delayed but greater toxicity of parathion in piperonyl butoxide pretreated mice.     

Antibodies to dehistonized chromatin of normal and fetal rat liver

Antisera to dehistonized adult or fetal rat liver chromatin were treated with electrophoretically separated chromosomal proteins of adult and fetal liver, Novikoff hepatoma and adult rat kidney. Both types of antisera reacted with numerous antigens in both tissue chromatins. However, immunoabsorption experiments have shown that while adult rat liver shared most of its nuclear antigens with other tissues, antisera to dehistonized fetal liver chromatins were more specific. In terms of antigenic specificity, the fetal rat liver and Novikoff hepatoma chromatins were similar; however, the former contained several antigens which could not be absorbed with Novikoff hepatoma chromatin.     

Somatomedin-C/insulin-like growth factor-I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues

Somatomedin-C or insulin-like growth factor I (Sm-C/IGF-I) and insulin-like growth factor II (IGF-II) have been implicated in the regulation of fetal growth and development. In the present study 32P-labeled complementary DNA probes encoding human and mouse Sm-C/IGF-I and human IGF-II were used in Northern blot hybridizations to analyse rat Sm-C/IGF-I and IGF-II mRNAs in poly(A+) RNAs from intestine, liver, lung, and brain of adult rats and fetal rats between day 14 and 17 of gestation. In fetal rats, all four tissues contained a major mRNA of 1.7 kilobases (kb) that hybridized with the human Sm-C/IGF-I cDNA and mRNAs of 7.5, 4.7, 1.7, and 1.2 kb that hybridized with the mouse Sm-C/IGF-I cDNA. Adult rat intestine, liver, and lung also contained these mRNAs but Sm-C/IGF-I mRNAs were not detected in adult rat brain. These findings provide direct support for prior observations that multiple tissues in the fetus synthesize immunoreactive Sm-C/IGF-I and imply a role for Sm-C/IGF-I in fetal development as well as postnatally. The abundance of a 7.5-kb Sm-C/IGF-I mRNA in poly(A+) RNAs from adult rat liver was 10-50-fold higher than in other adult rat tissues which provides further evidence that in the adult rat the liver is a major site of Sm-C/IGF-I synthesis and source of circulating Sm-C/IGF-I. Multiple IGF-II mRNAs of estimated sizes 4.7, 3.9, 2.2, 1.75, and 1.2 kb were observed in fetal rat intestine, liver, lung, and brain. The 4.7- and 3.9-kb mRNAs were the major hybridizing IGF-II mRNAs in all fetal tissues. Higher abundance of IGF-II mRNAs in rat fetal tissues compared with adult tissues supports prior hypotheses, based on serum IGF-II concentrations, that IGF-II is predominantly a fetal somatomedin. IGF-II mRNAs are present, however, in some poly(A+) RNAs from adult rat tissues. The brain was the only tissue in the adult rat where the 4.7- and 3.9-kb IGF-II mRNAs were consistently detected. Some samples of adult rat intestine contained the 4.7- and 3.9-kb IGF-II mRNAs and some samples of adult liver and lung contained the 4.7-kb mRNA. These findings suggest that a role for IGF-II in the adult rat, particularly in the central nervous system, cannot be excluded.     

Changes in parameters of lipoprotein metabolism during rat hepatic development

Maintenance of whole body cholesterol homeostasis is determined in part by the liver. Thus, changes in expression of hepatic parameters important in the regulation of cholesterol metabolism may play key roles in determining how homeostasis is maintained. The expression of hepatic lipoprotein uptake systems was studied during development using as a ligand very-low density lipoproteins rich in apolipoprotein E that had been obtained from hypercholesterolemic adult rats. These lipoproteins can serve as ligands for cell surface receptors recognizing apolipoproteins B and/or E. Uptake was lowest in freshly isolated fetal rat hepatocytes, increased substantially in hepatocytes from neonates and was intermediate in those from adults. Binding of these lipoproteins to liver membranes prepared from fetal, neonatal, suckling, weaned and adult rats was lowest in fetal preparations, while those from suckling, weaned and adult livers behaved similarly. Numbers of binding sites in neonatal liver membranes were similar to those in adult, but showed a different affinity. On the basis of this data, the ability of hepatocytes to recognize and remove apolipoprotein B/E-containing lipoproteins from the plasma appears to be a function of the differential expression or regulation of lipoprotein-uptake systems during development.     

Expression and Activation of Caspase-6 in Human Fetal and Adult Tissues

Caspase-6 is an effector caspase that has not been investigated thoroughly despite the fact that Caspase-6 is strongly activated in Alzheimer disease brains. To understand the full physiological impact of Caspase-6 in humans, we investigated Caspase-6 expression. We performed western blot analyses to detect the pro-Caspase-6 and its active p20 subunit in fetal and adult lung, kidney, brain, spleen, muscle, stomach, colon, heart, liver, skin, and adrenals tissues. The levels were semi-quantitated by densitometry. The results show a ubiquitous expression of Caspase-6 in most fetal tissues with the lowest levels in the brain and the highest levels in the gastrointestinal system. Caspase-6 active p20 subunits were only detected in fetal stomach. Immunohistochemical analysis of a human fetal embryo showed active Caspase-6 positive apoptotic cells in the dorsal root ganglion, liver, lung, kidney, ovary, skeletal muscle and the intestine. In the adult tissues, the levels of Caspase-6 were lower than in fetal tissues but remained high in the colon, stomach, lung, kidney and liver. Immunohistological analyses revealed that active Caspase-6 was abundant in goblet cells and epithelial cells sloughing off the intestinal lining of the adult colon. These results suggest that Caspase-6 is likely important in most tissues during early development but is less involved in adult tissues. The low levels of Caspase-6 in fetal and adult brain indicate that increased expression as observed in Alzheimer Disease is a pathological condition. Lastly, the high levels of Caspase-6 in the gastrointestinal system indicate a potential specific function of Caspase-6 in these tissues.     

Metabolism of [1-(14)C]arachidonic acid in ruminant tissues during the pre- and postnatal periods of development under in vitro conditions]

After in vitro incubation of liver, skeletal muscle and adipose tissues from the fetuses and adult cattle as well as of placenta tissue with [1-14C]arachidonic acid, about 90% of the radioactive label was found in the lipids, 10%-in the prostaglandins, and 0.1%-in 14CO2. Arachidonic acid was utilized for the synthesis of lipids and prostaglandins in the majority of fetal tissues in a much greater degree, whereas that in energy-linked process--in a smaller degree compared with adult cattle tissues. [1-14C]arachidonic acid metabolism in the placenta and liver of the given species proceeds much more intensely than that in the skeletal muscles and adipose tissue. In tissues of adult animals [1-14C]arachidonic acid is predominantly utilized for the synthesis of phospholipids, whereas that in fetal tissues is utilized for the synthesis of phospholipids, cholesterol, esters and triglycerides.     

Protein kinases in hepatoma, and adult and fetal liver of the rat. I. Subcellular distribution.

Comparison of the subcellular distribution of protein kinases in hepatoma, adult and fetal liver showed that nuclei of growing tissues contain a 3 to 5 times higher percentage of the activity in whole homogenate than nuclei of adult liver. The cytoplasmic protein kinase in hepatoma and fetal liver was stimulated by cAMP⁷ only half as much as that in adult liver. The nuclear activity was unresponsive to cAMP in all three tissues. Subfractionation of nuclei gave a final preparation that was more active with endogenous substrate than with added histone as phosphate acceptor. The hepatoma nuclei contained latent activity that could be unmasked in the presence of Triton X-100, those of adult and fetal liver did not. A partial resolution of the nuclear activity on DEAE-cellulose is reported.     

Three major forms of adenylate kinase from adult and fetal rat tissues.

The major enzymatic forms of adenylate kinase have been purified to homogeneity from fetal liver and adult brain of the rat. The two enzymes differ with respect to isoelectric points, Km (ATP), Km (AMP), and Ka (citrate). Antibody to adult liver adenylate kinase does not inhibit either enzyme, while entibody to adult skeletal muscle enzyme inhibits the brain enzyme but not the fetal liver enzyme. It is therefore probable that there are three major forms of adenylate kinases in fetal and adult rat tissues.     

Atrial natriuretic peptide in heart and specific binding in organs from fetal and newborn rats

To assess the possibility that atrial natriuretic peptide plays a role in salt and water balance during early mammalian development, we examined hearts from fetal and neonatal rates for the presence of this peptide and presumed target tissues for their ability to bind the hormone. Immunohistochemistry was used to localize and radioimmunoassay to quantify this peptide in heart. Immunoreactive atrial natriuretic peptide was visualized in the fetal heart on day 17.5 post-conception. It was distributed throughout the atrial appendages and free wall and, in ventricle, in the trabeculae carnae and chordae tendineae. The concentrations of immunoreactive atrial natriuretic peptide in atria of rats on day 19.5 post-conception were one-tenth of those in the adult. Levels of this peptide in fetal ventricle were low and virtually absent from the adult tissue. Specific binding of radiolabelled atrial natriuretic peptide measured by whole organ counting occurred in several organs from 19.5-day fetal and neonatal rats. A number of these tissues, including the kidney, ileum, adrenal, lung and liver, are targets for and/or bind the peptide in adult rats. Specific binding in these tissues was localized using autoradiography at anatomical sites similar to those in adult organs. Specific binding was also seen in fetal but not neonatal skin. In the kidney, binding was associated with immature as well as mature glomeruli. These findings support the proposition that atrial natriuretic peptide may function in the perinatal rat as it does in the adult and, in addition, may play a unique role during fetal life.     

Effects of methyl-deficient diets on methionine and homocysteine metabolism in the pregnant rat

Although the importance of methyl metabolism in fetal development is well recognized, there is limited information on the dynamics of methionine flow through maternal and fetal tissues and on how this is related to circulating total homocysteine concentrations. Rates of homocysteine remethylation in maternal and fetal tissues on days 11, 19, and 21 of gestation were measured in pregnant rats fed diets with limiting or surplus amounts of folic acid and choline at two levels of methionine and then infused with L-[1-(13)C,(2)H(3)-methyl]methionine. The rate of homocysteine remethylation was highest in maternal liver and declined as gestation progressed. Diets deficient in folic acid and choline reduced the production of methionine from homocysteine in maternal liver only in the animals fed a methionine-limited diet. Throughout gestation, the pancreas exported homocysteine for methylation within other tissues. Little or no methionine cycle activity was detected in the placenta at days 19 and 21 of gestation, but, during this period, fetal tissues, especially the liver, synthesized methionine from homocysteine. Greater enrichment of homocysteine in maternal plasma than placenta, even in animals fed the most-deficient diets, shows that the placenta did not contribute homocysteine to maternal plasma. Methionine synthesis from homocysteine in fetal tissues was maintained or increased when the dams were fed folate- and choline-deficient methionine-restricted diets. This study shows that methyl-deficient diets decrease the remethylation of homocysteine within maternal tissues but that these rates are protected to some extent within fetal tissues.     

Localisation of carcinoembryonic antigen in embryonic and fetal human tissues

Summary Carcinoembryonic antigen (CEA) was localized in various embryonic and fetal human tissues between 8 and 16 weeks of gestation as well as in the colorectal mucosa of older fetuses, newborns and adults. Among the embryonic tissues, CEA was always present in the esophagus, the gastric antrum, the duodenum and the rectum. CEA positive staining of bile cannaliculi of the liver was inconstant. All other embryonic tissues were CEA negative. During early fetal development CEA positive staining of the esophagus, antrum and duodenum was inconstant. However, the whole colon became intensively stained. An inconstant CEA specific staining was found in parts of the midgut and in the bile cannaliculi of the liver. The other organs remained CEA negative. Between the 17th week of gestation and birth, CEA staining pattern of the colorectal mucosa did not change. The staining intensity of late fetal colonic mucosa was similar to that of adult colonic mucosa.Deceased 20th August 1982     

Localisation of carcinoembryonic antigen in embryonic and fetal human tissues

Carcinoembryonic antigen (CEA) was localized in various embryonic and fetal human tissues between 8 and 16 weeks of gestation as well as in the colorectal mucosa of older fetuses, newborns and adults. Among the embryonic tissues, CEA was always present in the esophagus, the gastric antrum, the duodenum and the rectum. CEA positive staining of bile canaliculi of the liver was inconstant. All other embryonic tissues were CEA negative. During early fetal development, CEA positive staining of the esophagus, antrum and duodenum was inconstant. However, the whole colon became intensively stained. An inconstant CEA specific staining was found in parts of the midgut and in the bile canaliculi of the liver. The other organs remained CEA negative. Between the 17th week of gestation and birth, CEA staining pattern of the colorectal mucosa did not change. The staining intensity of late fetal colonic mucosa was similar to that of adult colonic mucosa.