Derivatives of 1,2,4-triazole imines acting as dual iNOS and tumor cell growth inhibitors.

A set of 4-(R2-imino)-3-mercapto-5-(R1)-4H-1,2,4-triazoles derivatives were synthesized, characterized and evaluated for their ability to inhibit nitric oxide (NO) production in PAM212 mouse keratinocytes, which led to the discovery and the subsequent evaluation of their growth inhibitory cytotoxic potency toward that same mouse cell line together with a number of human cells lines (PC3, HT-29 and HeLa). Some limited SAR could be established for both NO production inhibition potency and growth inhibition cytotoxicity. Noticeably, the compounds designed to be nitrofurantoin mimics were the most potent anti-neoplastic agents.

Interindividual Regulation of the Breast Cancer Resistance Protein/ABCG2 Transporter in Term Human Placentas.

The breast cancer resistance protein (BCRP/ABCG2) is a maternally-facing efflux transporter that regulates the placental disposition of chemicals. Transcription factors and gene variants are important regulatory factors that influence transporter expression. In this study, we sought to identify the genetic and transcriptional mechanisms underlying the interindividual expression of BCRP mRNA and protein across 137 term placentas from uncomplicated pregnancies. Placental expression of BCRP and regulatory transcription factor mRNAs was measured using multiplex-branched DNA analysis. BCRP expression and ABCG2 genotypes were determined using Western blot and Fluidigm Biomark genetic analysis, respectively. Placentas were obtained from a racially and ethnically diverse population, including Caucasian (33%), African American (14%), Asian (14%), Hispanic (15%), and mixed (16%) backgrounds, as well as unknown origins (7%). Between placentas, BCRP mRNA and protein varied up to 47-fold and 14-fold, respectively. In particular, BCRP mRNA correlated significantly with known transcription factor mRNAs, including nuclear factor erythroid 2-related factor 2 and aryl hydrocarbon receptor. Somewhat surprisingly, single-nucleotide polymorphisms (SNPs) in the ABCG2 noncoding regions were not associated with variation in placental BCRP mRNA or protein. Instead, the coding region polymorphism (C421A/Q141K) corresponded with 40%-50% lower BCRP protein in 421C/A and 421A/A placentas compared with wild types (421C/C). Although BCRP protein and mRNA expression weakly correlated (r = 0.25, P = 0.040), this relationship was absent in individuals expressing the C421A variant allele. Study results contribute to our understanding of the interindividual regulation of BCRP expression in term placentas and may help to identify infants at risk for increased fetal exposure to chemicals due to low expression of this efflux protein.

Genetic and Dietary Regulation of Glyburide Efflux by the Human Placental Breast Cancer Resistance Protein Transporter.

Glyburide is frequently used to treat gestational diabetes owing to its low fetal accumulation resulting from placental efflux by the breast cancer resistance protein (BCRP)/ABCG2 transporter. Here we sought to determine how exposure to the dietary phytoestrogen genistein and expression of a loss-of-function polymorphism in the ABCG2 gene (C421A) impacted the transport of glyburide by BCRP using stably transfected human embryonic kidney 293 (HEK) cells, human placental choriocarcinoma BeWo cells, and human placental explants. Genistein competitively inhibited the BCRP-mediated transport of (3)H-glyburide in both wild-type (WT) and C421A-BCRP HEK-expressing cells, with greater accumulation of (3)H-glyburide in cells expressing the C421A variant. In BeWo cells, exposure to genistein for 60 minutes increased the accumulation of (3)H-glyburide 30%-70% at concentrations relevant to dietary exposure (IC50 ∼180 nM). Continuous exposure of BeWo cells to genistein for 48 hours reduced the expression of BCRP mRNA and protein by up to 40%, which impaired BCRP transport activity. Pharmacologic antagonism of the estrogen receptor attenuated the genistein-mediated downregulation of BCRP expression, suggesting that phytoestrogens may reduce BCRP levels through this hormone receptor pathway in BeWo cells. Interestingly, genistein treatment for 48 hours did not alter BCRP protein expression in explants dissected from healthy term placentas. These data suggest that whereas genistein can act as a competitive inhibitor of BCRP-mediated transport, its ability to downregulate placental BCRP expression may only occur in choriocarcinoma cells. Overall, this research provides important mechanistic data regarding how the environment (dietary genistein) and a frequent genetic variant (ABCG2, C421A) may alter the maternal-fetal disposition of glyburide.

Investigation of anticholinergic and non-steroidal anti-inflammatory prodrugs which reduce chemically induced skin inflammation.

As part of a continuous effort to develop efficient counter measures against sulfur mustard injuries, several unique NSAID prodrugs have been developed and screened for anti-inflammatory properties. Presented herein are three classes of prodrugs which dually target inflammation and cholinergic dysfunction. Compounds 1-28 contain common NSAIDs linked either to choline bioisosteres or to structural analogs of acetylcholinesterase (AChE) inhibitors. These agents have shown utility as anti-vesicants and anti-inflammatory agents when screened in a mouse ear vesicant model (MEVM) against both 2-chloroethyl ethyl sulfide (CEES), a blistering agent, and 12-O-tetradecanoylphorbol-13-acetate (TPA), a common topical irritant. Many of the prodrugs have activity against CEES, with 5, 18, 22 and 27 reducing inflammation by more than 75% compared with a control. Compounds 12, 13, 15 and 22 show comparable activity against TPA. Promising activity in the MEVM is related to half-lives of NSAID release in plasma, moderate to high lipophilicity, and some degree of inhibition of AChE, a potential contributor to sulfur mustard-mediated tissue damage.

Decreased anti-inflammatory responses to vitamin D in neonatal neutrophils.

Neutrophil activity is prolonged in newborns, suggesting decreased exposure and/or responses to immunosuppressive modulators, such as 1,25-hydroxyvitamin D(3) (1,25-vit D(3)). We hypothesized that 1,25-vit D(3) suppresses neutrophil activation and that this response is impaired in newborns. Consistent with this, 1,25-vit D(3) decreased LPS-induced expression of macrophage inflammatory protein-1ß and VEGF in adult, but not neonatal, neutrophils. Expression of vitamin D receptor (VDR) and 25-hydroxyvitamin D(3)-1α-hydroxylase was reduced in neonatal, relative to adult neutrophils. Moreover, 1,25-vit D(3) induced VDR gene expression in activated adult, but not neonatal, neutrophils. 1,25-vit D(3) also suppressed expression of cyclooxygenase-2 and induced expression of 5-lipoxygenase in LPS-exposed adult neutrophils, while neonatal cells were not affected. 1,25-vit D(3) had no effect on respiratory burst in either adult or neonatal cells. Anti-inflammatory activity of vitamin D is impaired in neonatal neutrophils, and this may be due to decreased expression of VDR and 1α-hydroxylase. Insensitivity to 1,25-vit D(3) may contribute to chronic inflammation in neonates.

Low oxygen tension differentially regulates the expression of placental solute carriers and ABC transporters.

Low oxygen concentration, or hypoxia, is an important physiological regulator of placental function including chemical disposition. Here, we compared the ability of low oxygen tension to alter the expression of solute carriers (SLC) and ABC transporters in two human placental models, namely BeWo cells and term placental explants. We found that exposure to low oxygen concentration differentially regulates transporter expression in BeWo cells, including downregulation of ENT1, OATP4A1, OCTN2, BCRP, and MRP2/3/5, and upregulation of CNT1, OAT4, OATP2B1, SERT, SOAT, and MRP1. Similar upregulation of MRP1 and downregulation of MRP5 and BCRP were observed in explants, whereas uptake transporters were decreased or unchanged. Furthermore, a screening of transcriptional regulators of transporters revealed that hypoxia leads to a decrease in the mRNA levels of aryl hydrocarbon receptor, nuclear factor erythroid 2-related factor 2, and retinoid x receptor alpha in both human placental models. These data suggest that transporter expression is differentially regulated by oxygen concentration across experimental human placental models.

Inflammatory effects of phthalates in neonatal neutrophils.

Hospitalized infants are exposed to numerous devices containing the plasticizer di-(2-ethylhexyl) phthalate. Urinary levels of the phthalate metabolite, mono-(2-ethylhexyl) phthalate (MEHP), are markedly elevated in premature infants. Phthalates inactivate peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a nuclear transcription factor that mediates the resolution of inflammation, a process impaired in neonates. We speculate that this increases their susceptibility to MEHP, and this was analyzed. MEHP inhibited neutrophil apoptosis; neonatal cells were more sensitive than adult cells. In neonatal, but not in adult neutrophils, MEHP also inhibited chemotaxis, stimulated oxidative metabolism, and up-regulated expression of NADPH oxidase-1. In both adult and neonatal neutrophils, MEHP stimulated IL-1beta and VEGF production, whereas IL-8 production was stimulated only in adult cells. In contrast, MEHP-inhibited production of MIP-1beta by adult cells, and Regulated on Activation Normal T Cell Expressed and Secreted (RANTES) by neonatal neutrophils. The effects of MEHP on apoptosis and oxidative metabolism in neonatal cells were reversed by the PPAR-gamma agonist, troglitazone. Whereas troglitazone had no effect on MEHP-induced alterations in inflammatory protein or chemokine production, constitutive IL-8 and MIP-1beta production was reduced in adult neutrophils, and RANTES and MIP-1beta in neonatal cells. These findings suggest that neonatal neutrophils are more sensitive to phthalate-mediated inhibition of PPAR-gamma, which may be related to decreased anti-inflammatory signaling.

Mechanisms mediating reduced responsiveness of neonatal neutrophils to lipoxin A4.

Lipoxin A4 is an eicosanoid that plays a key role in the resolution of neutrophilic inflammation. In these studies, we investigated the hypothesis that responses to lipoxin A4 are impaired in neonates, relative to adults. Lipoxin A4 was found to inhibit chemotaxis and respiratory burst in adult neutrophils. In contrast, it had no effect on these activities in neonatal neutrophils. In addition, while lipoxin A4 augmented apoptosis in LPS-treated adult neutrophils, apoptosis in neonatal cells was not affected by lipoxin A4 alone or in combination with LPS. The biologic actions of anti-inflammatory eicosanoids are mediated, in part, via the transcription factor peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Expression of PPAR-gamma mRNA and its target gene, neutrophil gelatinase-associated lipocalin (NGAL), were significantly reduced in neonatal cells when compared with adult cells. Moreover, whereas treatment of adult neutrophils with lipoxin A4 increased PPAR-gamma expression, no effects were observed in neonatal cells. 5- and 15-lipoxygenase, enzymes required for the synthesis of lipoxin A4, were also reduced in neonatal neutrophils. These findings suggest that the anti-inflammatory activity of lipoxin A4 is impaired in neonatal neutrophils and that this is due, in part, to reduced PPAR-gamma signaling. This may contribute to diseases associated with chronic inflammation in neonates.

Localization of the placental BCRP/ABCG2 transporter to lipid rafts: Role for cholesterol in mediating efflux activity.

INTRODUCTION: The breast cancer resistance protein (BCRP/ABCG2) is an efflux transporter in the placental barrier. By transporting chemicals from the fetal to the maternal circulation, BCRP limits fetal exposure to a range of drugs, toxicants, and endobiotics such as bile acids and hormones. The purpose of the present studies was to 1) determine whether BCRP localizes to highly-ordered, cholesterol-rich lipid raft microdomains in placenta microvillous membranes, and 2) determine the impact of cholesterol on BCRP-mediated placental transport in vitro. METHODS: BCRP expression was analyzed in lipid rafts isolated from placentas from healthy, term pregnancies and BeWo trophoblasts by density gradient ultracentrifugation. BeWo cells were also tested for their ability to efflux BCRP substrates after treatment with the cholesterol sequestrant methyl-ß-cyclodextrin (MßCD, 5 mM, 1 h) or the cholesterol synthesis inhibitor pravastatin (200 µM, 48 h). RESULTS AND DISCUSSION: BCRP was found to co-localize with lipid raft proteins in detergent-resistant, lipid raft-containing fractions from placental microvillous membranes and BeWo cells. Treatment of BeWo cells with MßCD redistributed BCRP protein into higher density non-lipid raft fractions. Repletion of the cells with cholesterol restored BCRP localization to lipid raft-containing fractions. Treatment of BeWo cells with MßCD or pravastatin increased cellular retention of two BCRP substrates, the fluorescent dye Hoechst 33342 and the mycotoxin zearalenone. Repletion with cholesterol restored BCRP transporter activity. Taken together, these data demonstrate that cholesterol may play a critical role in the post-translational regulation of BCRP in placental lipid rafts.

Mono-(2-Ethylhexyl) Phthalate Promotes Pro-Labor Gene Expression in the Human Placenta.

Women exposed to phthalates during pregnancy are at increased risk for delivering preterm, but the mechanism behind this relationship is unknown. Placental corticotropin-releasing hormone (CRH) and cyclooxygenase-2 (COX-2) are key mediators of parturition and are regulated by the non-canonical NF-kB (RelB/p52) signaling pathway. In this study, we demonstrate that one of the major phthalate metabolites, mono-(2-ethylhexyl)-phthalate (MEHP), increased CRH and COX-2 mRNA and protein abundance in a dose-dependent manner in primary cultures of cytotrophoblast. This was coupled with an increase in nuclear import of RelB/p52 and its association with the CRH and COX-2 promoters. Silencing of NF-kB inducing kinase, a central signaling component of the non-canonical NF-kB pathway, blocked MEHP-induced upregulation of CRH and COX-2. These results suggest a potential mechanism mediated by RelB/p52 by which phthalates could prematurely induce pro-labor gene activity and lead to preterm birth.

Inhibition of interferon-gamma signaling by a mercurio-substituted dihydropsoralen in murine keratinocytes.

Psoralens and ultraviolet light A (PUVA) are used in the treatment of a variety of epidermal proliferative and inflammatory disorders. These compounds are known to intercalate and photo crosslink DNA. Specific receptor proteins for psoralens have also been identified. We describe a novel activity of a thiol reactive derivative, iodomercurio-4',5'-dihydrotrimethylpsoralen (iodomercurio-H2TMP) in keratinocytes. Without UVA, this psoralen was found to be an effective inhibitor of interferon-gamma (IFN-gamma)-signaling as measured by induction of nitric oxide biosynthesis (IC50 = 0.8 microM). This activity was increased (IC50 = 0.1 microM) when the cells were depleted of intracellular glutathione (GSH) with buthionine sulfoximine. In keratinocytes, IFN-gamma stimulates expression of inducible nitric oxide synthase (NOS2). Although iodomercurio-H2TMP did not alter NOS2 enzymatic activity, it blocked IFN-gamma-induced expression of NOS2 mRNA and protein, an effect that was enhanced in GSH-depleted cells. Iodomercurio-H2TMP was found to readily inhibit IFN-gamma signaling in transient transfection assays using NOS2 promoter/luciferase reporter constructs. NOS2 gene expression is known to require a variety of transcription factors including STAT-1, NF-kappaB and AP-1. Using mobility shift assays the psoralen, at concentrations that inhibit nitric oxide biosynthesis, had no effect on the DNA binding activity of STAT-1 or NF-kappaB. However, iodomercurio-H2TMP was found to suppress AP-1. These data indicate that iodomercurio-H2TMP acts at sulfhydryl-sensitive sites to inhibit NOS2. Moreover, this is dependent on early events in the IFN-gamma signal transduction pathway. Inhibition of AP-1 suggests that the psoralen functions by interfering with an important transcription factor that regulates expression of NOS2 in keratinocytes.

Association of prenatal perchlorate, thiocyanate, and nitrate exposure with neonatal size and gestational age.

BACKGROUND: Perchlorate and similar anions compete with iodine for uptake into the thyroid by the sodium iodide symporter (NIS). This may restrict fetal growth via impaired thyroid hormone production. METHODS: We collected urine samples from 107 pregnant women and used linear regression to estimate differences in newborn size and gestational age associated with increases in perchlorate, thiocyanate, nitrate, and perchlorate equivalence concentrations (PEC; measure of total NIS inhibitor exposure). RESULTS: NIS inhibitor concentrations were not associated with newborn weight, length, or gestational age. Each 2.62ng/µg creatinine increase in perchlorate was associated with smaller head circumference (0.32cm; 95% CI: -0.66, 0.01), but each 3.38ng/µg increase in PEC was associated with larger head circumference (0.48cm; -0.01, 0.97). CONCLUSIONS: These anions may have effects on fetal development (e.g. neurocognitive) that are not reflected in gross measures. Future research should focus on other abnormalities in neonates exposed to NIS inhibitors.

Effects of Wharton's jelly-derived mesenchymal stem cells on neonatal neutrophils.

BACKGROUND: Mesenchymal stem cells (MSCs) have been proposed as autologous therapy for inflammatory diseases in neonates. MSCs from umbilical cord Wharton's jelly (WJ-MSCs) are accessible, with high proliferative capacity. The effects of WJ-MSCs on neutrophil activity in neonates are not known. We compared the effects of WJ-MSCs on apoptosis and the expression of inflammatory, oxidant, and antioxidant mediators in adult and neonatal neutrophils. METHODS: WJ-MSCs were isolated, and their purity and function were confirmed by flow cytometry. Neutrophils were isolated from cord and adult blood by density centrifugation. The effects of neutrophil/WJ-MSC co-culture on apoptosis and gene and protein expression were measured. RESULTS: WJ-MSCs suppressed neutrophil apoptosis in a dose-dependent manner. WJ-MSCs decreased gene expression of NADPH oxidase-1 in both adult and neonatal neutrophils, but decreased heme oxygenase-1 and vascular endothelial growth factor and increased catalase and cyclooxygenase-2 in the presence of lipopolysaccharide only in adult cells. Similarly, generation of interleukin-8 was suppressed in adult but not neonatal neutrophils. Thus, WJ-MSCs dampened oxidative, vascular, and inflammatory activity by adult neutrophils, but neonatal neutrophils were less responsive. Conversely, Toll-like receptor-4, and cyclooxygenase-2 were upregulated in WJ-MSCs only in the presence of adult neutrophils, suggesting an inflammatory MSC phenotype that is not induced by neonatal neutrophils. CONCLUSION: Whereas WJ-MSCs altered gene expression in adult neutrophils in ways suggesting anti-inflammatory and antioxidant effects, these responses were attenuated in neonatal cells. In contrast, inflammatory gene expression in WJ-MSCs was increased in the presence of adult but not neonatal neutrophils. These effects should be considered in clinical trial design before WJ-MSC-based therapy is used in infants.

Cell-impermeant pyridinium derivatives of psoralens as inhibitors of keratinocyte growth.

Psoralens such as 8-methoxypsoralen and 4,5',8-trimethylpsoralen (TMP) are used in photochemotherapy for the treatment of a variety of epidermal proliferative diseases. Sequential treatments of the skin with psoralens plus ultraviolet light in the range of 320-400 nm (UVA light), referred to as PUVA therapy, results in the suppression of abnormal keratinocyte growth. With the recognition that the psoralens are phototoxic and carcinogenic, presumably due to their ability to intercalate into DNA and photo cross-link pyrimidine bases following UVA light activation, it is clear that the development of biologically active analogs lacking this activity would be of significant therapeutic benefit. Towards this goal we have characterized active 4'- and 5'-pyridinium derivatives of 4',5'-dihydro-TMP (H2TMP), a psoralen analog that does not form DNA cross-links. These analogs, which are charged at physiological pH and cannot penetrate cells, are unique in that they retain biological activity as inhibitors of keratinocyte cell growth when activated by UVA light. However, they do not appear to cross-link or damage DNA as determined by plasmid DNA unwinding and nicking experiments, in intact cells using fluorescent analysis of DNA unwinding assays, and by thymidine uptake studies. Reverse transcription-polymerase chain reaction and western blotting demonstrated that, unlike TMP and H2TMP, when activated by UVA light, the pyridinium derivatives were not inhibitors of transcription since interferon-gamma-inducible nitric oxide synthase mRNA and protein in the keratinocytes were unaffected. Taken together, our data suggest that uptake of the compounds by the cells and DNA cross-link formation are not required for growth inhibition. These findings further support the model that the cell membrane is an important target for the psoralens.

Mechanisms of growth inhibition in keratinocytes by mercurio-substituted 4',5'-dihydropsoralens.

Psoralens, together with ultraviolet light A (PUVA), are used in the treatment of epidermal proliferative disorders. Although these compounds can enter cells and photo cross-link DNA, lipids and proteins, including a specific membrane receptor, are also potential targets for the psoralens. To better elucidate the site of action of the psoralens, we have synthesized a family of 5'-mercurio-substituted derivatives of 4',5'-dihydropsoralen. These compounds are identified by their heavy metal content and can be used as a model to deliver thiol reactive psoralen derivatives into keratinocytes. The 5'-mercuriopsoralen derivatives were found to be effective inhibitors of keratinocyte growth without photoactivation. The most active compound, 4,8-dimethyl-5'-iodomercuriomethyl-4',5'-dihydropsoralen (IC50=10 microM), was also a potent photosensitizer (IC50=0.3 microM). Depletion of keratinocyte GSH with buthionine sulfoximine markedly increased their sensitivity to this analog, both with and without UVA light. In contrast, N-acetyl-L-cysteine partially protected the cells from growth inhibition, indicating that a sulfhydryl-sensitive site is growth limiting and that this target can be photoactivated. Iodomercurio-4',5'-dihydropsoralen was found to form adducts with GSH and cysteine, which were not active without UVA light. Thus, these adducts may also contribute to the photosensitization reactions of the parent compound. Using plasmid DNA unwinding assays, iodomercurio-4',5'-dihydropsoralen was also found to modify DNA, an activity that increased following UVA light treatment. This suggests that DNA damage may contribute to the actions of these psoralens. Taken together, our data demonstrate that there are multiple sites of action for mercuriopsoralens. These compounds may prove useful for understanding the mechanisms of psoralen-induced growth inhibition in the skin.

Regional expression of the BCRP/ABCG2 transporter in term human placentas.

The breast cancer resistance protein (BCRP, ABCG2) is an efflux transporter that removes xenobiotics that cross the placenta back to the maternal circulation, thereby limiting exposure of the fetus to drugs and chemicals. Currently, variability of BCRP expression within the placenta is not known. Ten placentas were collected from healthy women undergoing elective Cesarean sections at term. Villous samples were dissected in defined regions (medial, intermediate, and peripheral) and BCRP mRNA and protein were quantified. There were no regional differences in mRNA expression of housekeeping genes (GAPDH, RPL13a, PRL, 18S). GAPDH had the lowest correlation with BCRP Ct values and was used for BCRP mRNA normalization. No differences in placental BCRP mRNA and protein were observed among the sample sites (<20% variability). Sampling site does not affect the expression of BCRP, supporting the utility of single site sampling protocols to assess the interindividual regulation of this transporter in human placentas.

Effects of maternal exposure to phthalates and bisphenol A during pregnancy on gestational age.

OBJECTIVE: Phthalates and bisphenol A (BPA) are ubiquitous environmental toxicants, present in high concentrations in numerous consumer products. We hypothesized that maternal exposure to phthalates and BPA in pregnancy is associated with shortened gestation. METHODS: Urinary phthalate and BPA metabolites from 72 pregnant women were measured at the last obstetric clinic visit prior to delivery. Using linear regression models, we estimated the change in gestational age associated with each interquartile range (IQR) increase in phthalate and BPA metabolite concentration. RESULTS: IQR increases in urinary mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and BPA concentrations were associated with 4.2 and 1.1 d decreases in gestation, respectively. When stratified by gender, these alterations were found only in male infants. CONCLUSIONS: We conclude that MEHHP and BPA (free + glucuronide) are associated with reductions in gestation, with effects observed only in males. Our findings are consistent with the idea that these agents induce gender-specific alterations in signaling via PPAR-γ transcription factor, androgen precursors and/or inflammatory mediators during the initiation of labor.

Effects of bilirubin on neutrophil responses in newborn infants.

BACKGROUND: Newborns are susceptible to inflammatory diseases due to defects in clearing activated immune cells from tissues. Therefore, mechanisms have likely evolved to protect neonates from leukocyte-mediated cytotoxicity. Bilirubin has antioxidant activity, and it is possible that it also exerts effects on cellular immune responses in jaundiced infants. OBJECTIVES: We hypothesize that bilirubin increases expression of antioxidant genes and decreases production of inflammatory proteins in neonatal neutrophils. METHODS: Neutrophils were isolated from umbilical cord blood, and from adults for comparison, and treated with bilirubin (10-300 µmol/l, equivalent to unbound bilirubin 3-40 nmol/l), in the presence or absence of lipopolysaccharide (LPS). Expression of genes for antioxidant enzymes [superoxide dismutase (SOD), heme-oxygenase-1 (HO-1)] and heme-dependent enzymes involved in inflammation [NADPH oxidase-1 (NOX-1), cyclooxygenase-2 (COX-2)] was measured by PCR. Inflammatory cytokines were measured by bead array analysis using flow cytometry. RESULTS: We found that LPS induced production of interleukin (IL)-8, IL-1ß, and macrophage inhibitory protein-1ß (MIP-1ß). Bilirubin increased basal production of IL-8 and IL-1ß, but downregulated LPS-induced generation of IL-8 and MIP-1ß. It also upregulated SOD and HO-1 gene expression. We observed an unexpected bilirubin-induced increase in gene expression of NOX-1 in LPS-activated cells, and of COX-2 in both resting and activated cells. CONCLUSIONS: These findings suggest that bilirubin suppresses inflammation and increases antioxidant enzyme generation in activated neonatal neutrophils. The unexpected increases in NOX-1 and COX-2 expression may represent an early response, with physiologic effects mitigated by increased antioxidant activity. Further studies will be needed to define levels of bilirubin that optimize its protective effects, while minimizing potential inflammatory toxicity.

Mechanisms of oxidant generation by catalase.

The enzyme catalase converts solar radiation into reactive oxidant species (ROS). In this study, we report that several bacterial catalases (hydroperoxidases, HP), including Escherichia coli HP-I and HP-II also generate reactive oxidants in response to ultraviolet B light (UVB). HP-I and HP-II are identical except for the presence of NADPH. We found that only one of the catalases, HPI, produces oxidants in response to UVB light, indicating a potential role for the nucleotide in ROS production. This prompts us to speculate that NADPH may act as a cofactor regulating ROS generation by mammalian catalases. Structural analysis of the NADPH domains of several mammalian catalases revealed that the nucleotide is bound in a constrained conformation and that UVB irradiation induces NADPH oxidation and positional changes. Biochemical and kinetic analysis indicate that ROS formation by the enzyme is enhanced by oxidation of the cofactor. Conformational changes following absorption of UVB light by catalase NADPH have the potential to facilitate ROS production by the enzyme.

Influence of labor on neonatal neutrophil apoptosis, and inflammatory activity.

Neutrophil apoptosis is impaired in neonates, and this contributes to prolonged inflammation and tissue injury in infants after infection or trauma. In the present studies, we investigated whether labor generates mediators that further suppress apoptosis. We found that neutrophil apoptosis was reduced in neonates exposed to labor, when compared with infants delivered by cesarean section before labor. This was not due to alterations in caspase-3 or inhibitor of apoptosis protein-2 (IAP-2). In contrast, labor primed neutrophils to express tumor necrosis factor alpha (TNF-alpha), suggesting that proinflammatory mediators contribute to reduced apoptosis after labor. Eicosanoids generated via cyclooxygenase-2 (Cox-2) and lipoxygenase (Lox) also regulate neutrophil apoptosis. 15-Lox, which generates proapoptotic lipoxins, but not Cox-2, was greater in neutrophils before labor, relative to cells exposed to labor. Anti-inflammatory eicosanoids exert their effects in part via peroxisome proliferator-activated receptor gamma (PPAR-gamma). Expression of gelatinase-associated lipocalin and catalase, two markers of PPAR-gamma activity, were increased in neonatal neutrophils before labor, relative to cells exposed to labor. These findings suggest that the anti-inflammatory environment is maintained before labor, in part, by eicosanoids. Although increased neutrophil longevity after labor is important for host defense in the immediate newborn period, it may contribute to inflammatory or oxidative injury in susceptible infants.