Nitrate reductase activity of bacteria in saliva of term and preterm infants

The salivary glands of adults concentrate nitrate from plasma into saliva where it is converted to nitrite by bacterial nitrate reductases. Nitrite can play a beneficial role in adult gastrointestinal and cardiovascular physiology. When nitrite is swallowed, some of it is converted to nitric oxide (NO) in the stomach and may then exert protective effects in the gastrointestinal tract and throughout the body. It has yet to be determined either when newborn infants acquire oral nitrate reducing bacteria or what the effects of antimicrobial therapy or premature birth may be on the bacterial processing of nitrate to nitrite. We measured nitrate and nitrite levels in the saliva of adults and both preterm and term human infants in the early weeks of life. We also measured oral bacterial reductase activity in the saliva of both infants and adults, and characterized the species of nitrate reducing bacteria present. Oral bacterial conversion of nitrate to nitrite in infants was either undetectable or markedly lower than the conversion rates of adults. No measurable reductase activity was found in infants within the first two weeks of life, despite the presence of oral nitrate reducing bacteria such as Actinomyces odontolyticus, Veillonella atypica, and Rothia mucilaginosa. We conclude that relatively little nitrite reaches the infant gastrointestinal tract due to the lack of oral bacterial nitrate reductase activity. Given the importance of the nitrate-nitrite-NO axis in adults, the lack of oral nitrate-reducing bacteria in infants may be relevant to the vulnerability of newborns to hypoxic stress and gastrointestinal tract pathologies.     

Nitric oxide levels and antioxidant enzyme activities in jaundices of premature infants

Free radicals are effective in the genesis of several diseases in the neonatal period. This study aimed to show the relationship between serum bilirubin levels and plasma nitric oxide and the activity of enzymes in the erythrocyte such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in premature infants. In the study, 20 premature infants with newborn jaundice were included and the control group was formed by 15 premature infants without jaundice. Venous blood samples were taken from all neonates in the study and control groups on the first day of hospitalization. Plasma nitric oxide levels and activities of SOD, GSH-Px and CAT enzymes in the erythrocytes were investigated in these samples. Plasma nitric oxide and serum bilirubin levels were found to be significantly higher in the study group (47.4 +/- 7.25 micromol l(-1), 18.41 +/- 3.28 mg dl(-1), respectively) than those in the control group (33.46 +/- 6.43 micromol l(-1), 4.35 +/- 0.60 mg dl(-1), respectively; p < 0.001). In addition, erythrocyte SOD, GSH-Px and CAT enzyme activities (724 +/- 78.61, 673 +/- 90.5, 63 +/- 12.8 U g(-1) Hb, respectively) were found to be significantly lower in the study group than those in the control group (1208 +/- 129.04, 1097.6 +/- 75.8, 99.06 +/- 12.4 U g(-1) Hb, respectively, p < 0.001). It was concluded that in the aetiology of hyperbilirubinemia, neonatal erythrocytes and nitric oxide reactions are affected differently and that erythrocyte haemolysis caused as a result of these effects may play a role in the aetiopathogenesis of unconjugated hyperbilirubinemia. Haemolysis may also be seen because of the inadequacy of the protection by erythrocytes against the cytotoxic effects of free radicals resulting from the lack of antioxidant enzymes in these cells.     

Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease

Although the deleterious vasoconstrictive effects of cell-free, hemoglobin-based blood substitutes have been appreciated, the systemic effects of chronic hemolysis on nitric oxide bioavailability have not been considered or quantified. Central to this investigation is the understanding that nitric oxide reacts at least 1,000 times more rapidly with free hemoglobin solutions than with erythrocytes. We hypothesized that decompartmentalization of hemoglobin into plasma would divert nitric oxide from homeostatic vascular function. We demonstrate here that plasma from patients with sickle-cell disease contains cell-free ferrous hemoglobin, which stoichiometrically consumes micromolar quantities of nitric oxide and abrogates forearm blood flow responses to nitric oxide donor infusions. Therapies that inactivate plasma hemoglobin by oxidation or nitric oxide ligation restore nitric oxide bioavailability. Decompartmentalization of hemoglobin and subsequent dioxygenation of nitric oxide may explain the vascular complications shared by acute and chronic hemolytic disorders.     

Erythrocyte insulin binding in normal infants, children and adults

To establish normal insulin binding criteria, we studied the binding of insulin to erythrocytes from normal subjects of different ages. Insulin binding to cord erythrocytes and to erythrocytes from infants aged 2-7 days was significantly higher at tracer and physiological insulin concentrations than was binding to cells from children aged 1-15 years and adults. In infants aged 1-12 months the maximum insulin binding to erythrocytes was significantly higher than that to erythrocytes from children, and in addition, it correlated negatively with age. An increase in receptor concentration was found in cord erythrocytes whereas an increased receptor affinity for insulin was found in erythrocytes from infants. Insulin binding characteristics in erythrocytes from prepubertal and pubertal children were basically similar to those in women. Erythrocytes from men bound significantly higher amounts of insulin than did those from women. This difference was associated with changes in receptor affinity for insulin. There was no correlation between the insulin binding characteristics and the circulating concentration of insulin or C-peptide. The increased erythrocyte insulin binding at birth persisted over the neonatal period. There was an overall negative correlation between the maximum insulin binding and age in the subjects studied, but the major decrease in erythrocytes insulin binding occurred during the first year of life past the neonatal period. These observations stress the importance of using age-matched controls in studies on erythrocyte insulin binding in disease states.     

Iron release, superoxide production and binding of autologous IgG to band 3 dimers in newborn and adult erythrocytes exposed to hypoxia and hypoxia-reoxygenation

Iron is released in a desferrioxamine (DFO)-chelatable form when erythrocytes are challenged by an oxidative stress. The release is increased when an accelerated removal of erythrocytes occurs such as in perinatal period, in which iron release is greater in hypoxic than in non-hypoxic newborns. This suggests that an hypoxic environment at birth promotes iron release. To test this possibility, iron release in a model of hypoxia, hypoxia-reoxygenation and normoxia was studied in newborn and adult erythrocytes. In newborn erythrocytes, hypoxia induced a much greater iron release compared to an equal period of normoxia. In adult erythrocytes, hypoxia also induced a greater iron release as compared to normoxia, but it was much lower than that seen with newborn erythrocytes. Methemoglobin (MetHb) formation roughly paralleled iron release. The phenylhydrazine-promoted superoxide anion (O(2)?(-)) production was greater with normoxic but lower with hypoxic erythrocytes from newborns as compared to that from adults. This discrepancy between iron release and O(2)?(-) production may be explained by the shift towards MetHb in hemoglobin autoxidation. Iron diffusion out of the erythrocytes was much higher with hypoxic erythrocytes from newborns as compared to that from adults. Also the binding of autologous IgG to band 3 dimers (AIgGB) is much greater with hypoxic erythrocytes from newborns as compared to that from adults, suggesting that the level of iron release is related to the extent of band 3 clustering and that hypoxia accelerates removal of erythrocytes from bloodstream in in vivo condition.     

Effect of ascorbic acid on production of nitric oxide by leukocytes

The effect of ascorbic acid on suspensions of blood formic elements was studied by the ESR method. It was shown that incubation of a suspension of formic blood elements in the presence of ascorbic acid leads to the appearance of nitric oxide, which is produced by leukocytes and partially probably by thrombocytes. The formation of nitric oxide is evidenced by the appearance of nitrosyl complexes heme-NO. In this case, hemoglobin of erythrocytes serves as a natural trap for nitric oxide.     

Diagnostic Accuracy of S100B Urinary Testing at Birth in Full-Term Asphyxiated Newborns to Predict Neonatal Death

Background

Neonatal death in full-term infants who suffer from perinatal asphyxia (PA) is a major subject of investigation, since few tools exist to predict patients at risk of ominous outcome. We studied the possibility that urine S100B measurement may identify which PA-affected infants are at risk of early postnatal death.

Methodology/Principal Findings

In a cross-sectional study between January 1, 2001 and December 1, 2006 we measured S100B protein in urine collected from term infants (n = 132), 60 of whom suffered PA. According to their outcome at 7 days, infants with PA were subsequently classified either as asphyxiated infants complicated by hypoxic ischemic encephalopathy with no ominous outcome (HIE Group; n = 48), or as newborns who died within the first post-natal week (Ominous Outcome Group; n = 12). Routine laboratory variables, cerebral ultrasound, neurological patterns and urine concentrations of S100B protein were determined at first urination and after 24, 48 and 96 hours. The severity of illness in the first 24 hours after birth was measured using the Score for Neonatal Acute Physiology-Perinatal Extension (SNAP-PE). Urine S100B levels were higher from the first urination in the ominous outcome group than in healthy or HIE Groups (p<0.001 for all), and progressively increased. Multiple logistic regression analysis showed a significant correlation between S100B concentrations and the occurrence of neonatal death. At a cut-off >1.0 µg/L S100B had a sensitivity/specificity of 100% for predicting neonatal death.

Conclusions/Significance

Increased S100B protein urine levels in term newborns suffering PA seem to suggest a higher risk of neonatal death for these infants.     

High constitutional nitrate status in young cattle.

Nitrate or nitrite can be ingested or endogenously produced from nitric oxide. They can cause intoxication and are of general concern for health because they relate to various diseases. Our goal was to study ontogenetic and nutritional effects on the nitrate+nitrite (NOx-) status in cattle, particularly calves. NOx- concentration in blood plasma, cerebrospinal fluid, saliva, and urine was measured based on nitrate conversion by added nitrate reductase to nitrite, which was then determined by the Griess reaction. Concentrations of nitrate were the result of the difference between NOx- and nitrite values. Nitrate in blood plasma, saliva and urine was > or =97% and in cerebrospinal fluid of calves was approximately 35% of NOx-. Preprandial plasma NOx- in calves born after shortened or normal lengths of pregnancy (277 and 290 days) was 470 and 830 micromol/l, respectively, decreased within 4-7 days to 40-60 micromol/l, remained in this range up to 4 months, was < or =5 micromol/l in heifers and no longer measurable in 3-8-year-old cows. Cerebrospinal NOx- in 8-day-old calves was 14 micromol/l and approximately 11-fold lower than in blood plasma. Salivary NOx- decreased postnatally from 600 to 200 micromol/l at 2 days and to 25 micromol/l at 4 weeks. Urinary NOx- excretion decreased from 125 or 16 micromol/l per kg x 24 h in 5-day-old calves to 45 or 8 micromol/kg x 24 h between 10 and 115 days of life and was undetectable in urine of heifers and cows. Feeding neonatal calves no or variable amounts of colostrum, delaying colostrum intake by 24 h after birth or feeding at different feeding intensity had no effect on the NOx- status. In conclusion, the high plasma, salivary and urinary NOx- concentrations especially in newborn calves, ingesting but insignificant amounts of nitrite or nitrate, indicated marked endogenous formation of nitrate, which decreased with age. The high nitrate status may contribute to enhanced susceptibility of young calves to exogenous nitrite+nitrite ingestion.     

Formation of nitric oxide hemoglobin in erythrocytes co-cultured with alveolar macrophages taken from bleomycin treated rats.

Alveolar macrophages, taken from rats treated with a single intratracheal dose of bleomycin, release reactive nitrogen intermediates in the form of nitric oxide which are cytostatic to murine leukemia L1210 cells. When cultured in the presence of erythrocytes the cytostatic activity of alveolar macrophages was inhibited which corresponded with an increase in nitrosylated hemoglobin content when compared with erythrocytes cultured alone. These results suggest that erythrocytes inhibit alveolar macrophage cytostatic activity by preventing reactive nitrogen intermediates from reaching target cells because the hemoglobin serves as a sink for reactive nitrogen intermediates in the form of nitric oxide.     

Influence of maternal insulin-dependent diabetes mellitus on neonatal morbidity.

OBJECTIVE: To compare the neonatal morbidity rates (corrected for gestational age at delivery and method of delivery) among infants of women with insulin-dependent diabetes mellitus and those of women without diabetes. DESIGN: Historical cohort analysis. SETTING: Tertiary care centre. PATIENTS: All liveborn infants of women with insulin-dependent diabetes mellitus (IDM group) born between Jan. 1, 1980, and Dec. 31, 1989, each matched for gestational age at delivery, method of delivery and year of birth with two newborns of women without diabetes (control group). MAIN OUTCOME MEASURES: Neonatal respiratory distress, jaundice, hypoglycemia, polycythemia, hypocalcemia, intraventricular hemorrhage, seizure and macrosomia. RESULTS: There were 230 infants in the IDM group and 460 in the control group. Compared with the control group the IDM group had significantly higher incidence rates of glucose infusion (odds ratio [OR] 5.38), birth weight above the 90th percentile (OR 4.15) and neonatal jaundice (OR 1.94). No significant difference was found in the incidence rate of respiratory distress, polycythemia or hypocalcemia. The maternal serum hemoglobin A (HbA) level was not significantly related to birth weight, and neither the serum HbA level nor the presence of macrosomia was predictive of neonatal morbidity. Nearly 25% of the infants in the IDM group were born before 37 weeks'' gestation; 48.2% of these were delivered early because of maternal hypertension. CONCLUSIONS: Neonatal morbidity in infants of women with diabetes is determined more by gestational age at delivery than by the maternal diabetes. Within the limits obtained in this study the degree of control of the diabetes does not seem to affect neonatal morbidity.     

Oxidation of iron-nitrosyl-hemoglobin by dehydroascorbic acid releases nitric oxide to form nitrite in human erythrocytes

The reaction of deoxyhemoglobin with nitric oxide (NO) or nitrite ions (NO 2 (-)) produces iron-nitrosyl-hemoglobin (HbNO) in contrast to the reaction with oxyhemoglobin, which produces methemoglobin and nitrate (NO 3 (-)). HbNO has not been associated with the known bioactivities of NO. We hypothesized that HbNO in erythrocytes could be an important source of bioactive NO/nitrite if its oxidation was coupled to the ascorbic acid (ASC) cycle. Studied by absorption and electron paramagnetic resonance (EPR) spectroscopy, DHA oxidized HbNO to methemoglobin and liberated NO from HbNO as determined by chemiluminescence. Both DHA and ascorbate free radical (AFR), the intermediate between ASC and DHA, enhanced NO oxidation to nitrite, but not nitrate; nor did either oxidize nitrite to nitrate. DHA increased the basal levels of nitrite in erythrocytes, while the reactions of nitrite with hemoglobin are slow. In erythrocytes loaded with HbNO, HbNO disappeared after DHA addition, and the AFR signal was detected by EPR. We suggest that the ASC-AFR-DHA cycle may be coupled to that of HbNO-nitrite and provide a mechanism for the endocrine transport of NO via hemoglobin within erythrocytes, resulting in the production of intracellular nitrite. Additionally, intracellular nitrite and nitrate seem to be largely generated by independent pathways within the erythrocyte. These data provide a physiologically robust mechanism for erythrocytic transport of NO bioactivity allowing for hormone-like properties.     

Carbon monoxide excretion as an index of bilirubin production in rhesus monkeys

The role of increased heme catabolism in neonatal hyperbilirubinemia was investigated in rhesus (Macaca mulatta) neonates through the measurement of carbon monoxide excretion rates (VECO), blood carboxyhemoglobin content (HbCO), and plasma bilirubin concentrations. Neonatal values were compared to those of adult rhesus monkeys. These indices of bilirubin production responded appropriately to administration of NEM-damaged erythrocytes and tin protoporphyrin. Our results indicate that VECO measurements are a valid index of changes in bilirubin production in the newborn rhesus monkey.     

Hemoglobin-nitric oxide cooperativity: is NO the third respiratory ligand?

Through its cooperative binding mechanism, hemoglobin is an effective transporter of oxygen and carbon dioxide. Although data have recently been presented suggesting otherwise, the rate at which nitric oxide binds to hemoglobin is not cooperative. On the other hand, the rate at which nitric oxide dissociates from hemoglobin is cooperative so that, similar to the case of oxygen, the cooperativity in equilibrium ligand binding is manifested in the dissociation rate rather than the association rate. Two general factors that diminish the likelihood that hemoglobin transports nitric oxide are the slow dissociation rate of nitric oxide from hemoglobin and the very fast hemoglobin oxidation reaction, which converts nitric oxide to the inert molecule nitrate. Despite these factors the possibility that NO is delivered by hemoglobin under certain conditions or through more complicated mechanisms needs further study.     

Ultrastructure and peroxidase cytochemistry of normal human leukocytes at birth

At birth, differential and white blood cell counts of normal newborn infants are strikingly different from those of adults in that the number of leukocytes is increased and immature cells course through the circulation. In this study, our intent was to examine normal neonatal cord blood by electron microscopy and peroxidase cytochemistry to determine whether any detectable differences exist in the leukocytes of neonatal and adult blood. This investigation was undertaken because newborn infants have an increased susceptibility to infection, and alterations in phagocyte function have been implicated as the cause. Cord blood was found to contain mature leukocytes of all kinds, similar in ultrastructure and peroxidase localization to those of adults. Moreover, as indicated earlier by light microscopy, immature forms (normally found only in adult bone marrow) were present in the blood of newborns. We found that nearly all cell lines were represented in the neonatal circulation by such developmental forms as promyelocytes, myelocytes, promonocytes, erythroblasts, megakaryocytes, rare unidentifiable blasts, and dividing cells—all resembling their counterparts in adult bone marrow. With the techniques used here, neonatal leukocytes were similar to those of the adult in ultrastructure and peroxidase localization, although some had been mobilized into the blood in a remarkably immature state. This study, the first of its kind, will serve as a helpful background for future investigations of acquired, genetic or neoplastic leukocyte abnormalities which may be discovered at birth.     

Changes of nitric oxide,carbon monoxide and oxidative stress in term infants at birth

The higher risk of respiratory problem in infants delivered by elective caesarean section in comparison with vaginally born infants may be favoured by lower level of nitric oxide (NO) and carbon monoxide (CO) and higher oxidative stress in infants born by caesarean section. We studied healthy term infants born by vaginal delivery or by elective caesarean section. Nitric oxide, CO, guanosine 3–5 cyclic monophosphate, total hydroperoxide and advanced oxidation protein products (AOPP) were measured at birth and 48–72 h of life. Nitric oxide, CO and cGMP were lower at birth and at 48–72 h of life in infants born by elective caesarean delivery. Total hydroperoxide and AOPP levels were similar in the two groups and increased from birth to 48–72 h of life. In conclusion, nitric oxide and CO concentrations were higher in term infants vaginally born than in infants born by elective caesarean section and decreased from birth to 48–72 h of life. The mode of delivery did not affect the oxidative stress which increases from birth to 48–72 h of life.     

Changes of nitric oxide, carbon monoxide and oxidative stress in term infants at birth

The higher risk of respiratory problem in infants delivered by elective caesarean section in comparison with vaginally born infants may be favoured by lower level of nitric oxide (NO) and carbon monoxide (CO) and higher oxidative stress in infants born by caesarean section. We studied healthy term infants born by vaginal delivery or by elective caesarean section. Nitric oxide, CO, guanosine 3-5 cyclic monophosphate, total hydroperoxide and advanced oxidation protein products (AOPP) were measured at birth and 48-72 h of life. Nitric oxide, CO and cGMP were lower at birth and at 48-72 h of life in infants born by elective caesarean delivery. Total hydroperoxide and AOPP levels were similar in the two groups and increased from birth to 48-72 h of life. In conclusion, nitric oxide and CO concentrations were higher in term infants vaginally born than in infants born by elective caesarean section and decreased from birth to 48-72 h of life. The mode of delivery did not affect the oxidative stress which increases from birth to 48-72 h of life.     

Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite

Tyrosine nitration is a widely used marker of peroxynitrite (ONOO(-)) produced from the reaction of nitric oxide with superoxide. Pfeiffer and Mayer (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) reported that superoxide produced from hypoxanthine plus xanthine oxidase in combination with nitric oxide produced from spermine NONOate did not nitrate tyrosine at neutral pH. They suggested that nitric oxide and superoxide at neutral pH form a less reactive intermediate distinct from preformed alkaline peroxynitrite that does not nitrate tyrosine. Using a stopped-flow spectrophotometer to rapidly mix potassium superoxide with nitric oxide at pH 7.4, we report that an intermediate spectrally and kinetically identical to preformed alkaline cis-peroxynitrite was formed in 100% yield. Furthermore, this intermediate nitrated tyrosine in the same yield and at the same rate as preformed peroxynitrite. Equivalent concentrations of nitric oxide under aerobic conditions in the absence of superoxide did not produce detectable concentrations of nitrotyrosine. Carbon dioxide increased the efficiency of nitration by nitric oxide plus superoxide to the same extent as peroxynitrite. In experiments using xanthine oxidase as a source of superoxide, tyrosine nitration was substantially inhibited by urate formed from hypoxanthine oxidation, which was sufficient to account for the lack of tyrosine nitration previously reported. We conclude that peroxynitrite formed from the reaction of nitric oxide with superoxide at physiological pH remains an important species responsible for tyrosine nitration in vivo.     

Plasma S-nitrosothiol status in neonatal calves: ontogenetic associations with tissue-specific S-nitrosylation and nitric oxide synthase

Neonatal cattle and in part neonates of other species have manyfold higher plasma concentrations of nitrite plus nitrate than mature cows and subjects of other species, suggesting an enhanced and needed activation of the nitric oxide (NO) axis at birth. While the biological half-life of NO is short (<1 sec), its functionality can be prolonged, and in many regards more discretely modulated, when it reacts with low-molecular-weight and protein-bound thiols to form S-nitrosothiols (RSNO), from which NO subsequently can be rereleased. We used the calf as a model to test the hypothesis that plasma concentrations of RSNO are elevated at birth in mammals, correlate with ascorbate and urate levels, are selectively generated in critical tissue beds, and are generated in a manner temporally coincident with changes in tissue levels of active NO synthases (NOS). Plasma concentrations of RSNO, ascorbate, and urate were highest immediately after birth (Day 0), dropped >50% on Day 1, and gradually decreased over time, reaching a nadir in mature cattle. Albumin and immunoglobulin G were identified as major plasma RSNO. The presence of S-nitrosocysteine (SNC, a validated marker for S-nitrosylated proteins), inducible NOS (iNOS), and activated endothelial NOS (eNOS phosphorylated at Ser1177) in different tissues was analyzed by immunohistochemistry in another group of similar-aged calves. SNC, iNOS, and phosphorylated eNOS were detected in liver and ileum at the earliest timepoint of sampling (4 hrs after birth), increased between 4 and 24 hrs, and then declined to near-nondetectable levels by 2 weeks of life. Our data show that the neonatal period in the bovine species is characterized by highly elevated and coordinated NO-generating and nitrosylation events, with the ontogenetic changes occurring in iNOS and eNOS contents in key tissues as well as RSNO products and associated antioxidant markers.     

Essential fatty acids in erythrocyte phospholipids during pregnancy and at delivery in mothers and their neonates: comparison with plasma phospholipids

Evidence that the essential fatty acid (EFA) status during pregnancy and at birth may not be optimal is mainly based on fatty acid profiles of maternal and neonatal plasma phospholipids. However, erythrocyte phospholipids may be more reliable than plasma phospholipids to reflect the EFA status of an individual. Therefore, the present study compares the levels of EFA and of their derivatives (LCPUFA) in erythrocyte and plasma phospholipids collected during pregnancy and at delivery of 184 women and of their infants at birth. In general, the relative concentrations of erythrocyte and plasma phospholipid fatty acids (% of total fatty acids) were strongly correlated, but not at early pregnancy. The overall changes in fatty acid concentrations during pregnancy were qualitatively comparable between erythrocytes and plasma, although the comparability became less towards the end of pregnancy. The changes in absolute amounts (mg/l) of fatty acids in erythrocyte and plasma phospholipids also compare quite well till 32 weeks of gestation, but not thereafter. Most maternal-neonatal differences in relative fatty acid concentrations are qualitatively comparable for erythrocyte and plasma phospholipids. However, significant differences were observed for the absolute amounts of arachidonic and docosahexaenoic acids. No matter these differences, plasma and erythrocyte phospholipids seem equally suitable to reliably quantify the more functional EFA and LCPUFA status based on fatty acid ratios. Correlations between neonatal and maternal fatty acid values at delivery/birth are highly significant in erythrocyte as well as plasma phospholipids. Neonatal erythrocyte (but not plasma) values also correlated strongly with maternal values at early pregnancy. Therefore, the neonatal EFA and LCPUFA status might be predicted on the basis of EFA and LCPUFA concentrations of maternal erythrocyte phospholipids at early pregnancy.     

Protection of Human Keratinocyte mtDNA by Low-Level Nitric Oxide

This study was designed to evaluate the DNA damaging effects of nitric oxide and to determine whether the endogenous generation of nitric oxide at low levels in the cell exerts a protective effect against this damage. Damage to mitochondrial and nuclear DNA in normal human epidermal keratinocytes (NHEK) was assessed after treatment of these cells with varying concentrations of S-nitroso-N-acetylpenicillamine, which decomposes to release nitric oxide. The results showed that mitochondrial DNA was more vulnerable to nitric oxide-induced damage than was a similarly sized fragment of the beta-globin gene. To evaluate the effects on DNA damage by pretreatment of cells with low-levels of nitric oxide, NHEK cells were treated with the prodrug V-PYRRO/NO. This agent is metabolized inside these cells and releases small quantities of nitric oxide. The cells then were exposed to damaging amounts of nitric oxide produced by S-nitroso-N-acetylpenicillamine. The results of these studies showed that pretreatment of NHEK cells with V-PYRRO/NO attenuated the mtDNA damage and loss of cell viability produced by exposure to S-nitroso-N-acetylpenicillamine.