T-helper 1-type cytokines induce apoptosis and loss of HER-family oncodriver expression in murine and human breast cancer cells.

A recent neoadjuvant vaccine trial for early breast cancer induced strong Th1 immunity against the HER-2 oncodriver, complete pathologic responses in 18% of subjects, and for many individuals, dramatically reduced HER-2 expression on residual disease. To explain these observations, we investigated actions of Th1 cytokines (TNF-α and IFN-γ) on murine and human breast cancer cell lines that varied in the surface expression of HER-family receptor tyrosine kinases. Breast cancer lines were broadly sensitive to the combination of IFN-γ and TNF-α, as evidenced by lower metabolic activity, lower proliferation, and enhanced apoptosis, and in some cases a reversible inhibition of surface expression of HER proteins. Apoptosis was accompanied by caspase-3 activation. Furthermore, the pharmacologic caspase-3 activator PAC-1 mimicked both the killing effects and HER-2-suppressive activities of Th1 cytokines, while a caspase 3/7 inhibitor could prevent cytokine-induced HER-2 loss. These studies demonstrate that many in vivo effects of vaccination (apparent tumor cell death and loss of HER-2 expression) could be replicated in vitro using only the principle Th1 cytokines. These results are consistent with the notion that IFN-γ and TNF-α work in concert to mediate many biological effects of therapeutic vaccination through the induction of a caspase 3-associated cellular death mechanism.

The early infant gut microbiome varies in association with a maternal high-fat diet.

BACKGROUND: Emerging evidence suggests that the in utero environment is not sterile as once presumed. Work in the mouse demonstrated transmission of commensal bacteria from mother to fetus during gestation, though it is unclear what modulates this process. We have previously shown in the nonhuman primate that, independent of obesity, a maternal high-fat diet during gestation and lactation persistently shapes the juvenile gut microbiome. We therefore sought to interrogate in a population-based human longitudinal cohort whether a maternal high-fat diet similarly alters the neonatal and infant gut microbiome in early life. METHODS: A representative cohort was prospectively enrolled either in the early third trimester or intrapartum (n = 163), with a subset consented to longitudinal sampling through the postpartum interval (n = 81). Multiple body site samples, including stool and meconium, were collected from neonates at delivery and by 6 weeks of age. A rapid dietary questionnaire was administered to estimate intake of fat, added sugars, and fiber over the past month (National Health and Examination Survey). DNA was extracted from each infant meconium/stool sample (MoBio) and subjected to 16S rRNA gene sequencing and analysis. RESULTS: On average, the maternal dietary intake of fat ranged from 14.0 to 55.2 %, with an average intake of 33.1 % (σ = 6.1 %). Mothers whose diets significantly differed from the mean (±1 standard deviation) were separated into two distinct groups, a control group (n = 13, µ = 24.4 %) and a high-fat group (n = 13, µ = 43.1 %). Principal coordinate analysis revealed that the microbiome of the neonatal stool at birth (meconium) clustered differently by virtue of maternal gestational diet (PERMANOVA p = 0.001). LEfSe feature selection identified several taxa that discriminated the groups, with a notable relative depletion of Bacteroides in the neonates exposed to a maternal high-fat gestational diet (Student's t-test, p < 0.05) that persisted to 6 weeks of age. CONCLUSIONS: Similar to the primate, independent of maternal body mass index, a maternal high-fat diet is associated with distinct changes in the neonatal gut microbiome at birth which persist through 4-6 weeks of age. Our findings underscore the importance of counseling pregnant mothers on macronutrient consumption during pregnancy and lactation.

Progressive loss of anti-HER2 CD4+ T-helper type 1 response in breast tumorigenesis and the potential for immune restoration.

Genomic profiling has identified several molecular oncodrivers in breast tumorigenesis. A thorough understanding of endogenous immune responses to these oncodrivers may provide insights into immune interventions for breast cancer (BC). We investigated systemic anti-HER2/neu CD4+ T-helper type-1 (Th1) responses in HER2-driven breast tumorigenesis. A highly significant stepwise Th1 response loss extending from healthy donors (HD), through HER2pos-DCIS, and ultimately to early stage HER2pos-invasive BC patients was detected by IFNγ ELISPOT. The anti-HER2 Th1 deficit was not attributable to host-level T-cell anergy, loss of immune competence, or increase in immunosuppressive phenotypes (Treg/MDSCs), but rather associated with a functional shift in IFNγ:IL-10-producing phenotypes. HER2high, but not HER2low, BC cells expressing IFNγ/TNF-α receptors were susceptible to Th1 cytokine-mediated apoptosis in vitro, which could be significantly rescued by neutralizing IFNγ and TNF-α, suggesting that abrogation of HER2-specific Th1 may reflect a mechanism of immune evasion in HER2-driven tumorigenesis. While largely unaffected by cytotoxic or HER2-targeted (trastuzumab) therapies, depressed Th1 responses in HER2pos-BC patients were significantly restored following HER2-pulsed dendritic cell (DC) vaccinations, suggesting that this Th1 defect is not "fixed" and can be corrected by immunologic interventions. Importantly, preserved anti-HER2 Th1 responses were associated with pathologic complete response to neoadjuvant trastuzumab/chemotherapy, while depressed responses were observed in patients incurring locoregional/systemic recurrence following trastuzumab/chemotherapy. Monitoring anti-HER2 Th1 reactivity following HER2-directed therapies may identify vulnerable subgroups at risk of clinicopathologic failure. In such patients, combinations of existing HER2-targeted therapies with strategies to boost anti-HER2 CD4+ Th1 immunity may decrease the risk of recurrence and thus warrant further investigation.

Mitochondrial DNA sequence variation is largely conserved at birth with rare de novo mutations in neonates.

OBJECTIVE: Mitochondrial DNA (mtDNA) encodes the proteins of the electron transfer chain to produce adenosine triphosphate through oxidative phosphorylation, and is essential to sustain life. mtDNA is unique from the nuclear genome in so much as it is solely maternally inherited (non-mendelian patterning), and shows a relatively high rate of mutation due to the absence of error checking capacity. While it is generally assumed that most new mutations accumulate through the process of heteroplasmy, it is unknown whether mutations initiated in the mother are inherited, occur in utero, or occur and accumulate early in life. The purpose of this study is to examine the maternally heritable and de novo mutation rate in the fetal mtDNA through high-fidelity sequencing from a large population-based cohort. STUDY DESIGN: Samples were obtained from 90 matched maternal (blood) and fetal (placental) pairs. In addition, a smaller cohort (n = 5) of maternal (blood), fetal (placental), and neonatal (cord blood) trios were subjected to DNA extraction and shotgun sequencing. The whole genome was sequenced on the Illumina HiSeq platform (Illumina Inc., San Diego, CA), and haplogroups and mtDNA variants were identified through mapping to reference mitochondrial genomes (NC_012920). RESULTS: We observed 665 single nucleotide polymorphisms and 82 insertions-deletions variants identified in the cohort at large. We achieved high sequencing depth of the mtDNA to an average depth of 65X (range, 20-171X) coverage. The proportions of haplogroups identified in the cohort are consistent with the patient's self-identified ethnicity (>90% Hispanic), and all maternal-fetal pairs mapped to the identical haplogroup. Only variants from samples with average depth >20X and allele frequency >1% were included for further analysis. While the majority of the maternal-fetal pairs (>90%) demonstrated identical variants at the single nucleotide level, we observed rare mitochondrial single nucleotide polymorphism discordance between maternal and fetal mitochondrial genomes. CONCLUSION: In this first in-depth sequencing analysis of mtDNA from maternal-fetal pairs at the time of birth, a low rate of de novo mutations appears in the fetal mitochondrial genome. This implies that these mutations likely arise from the maternal heteroplasmic pool (eg, in the oocyte), and accumulate later in the offspring's life. These findings have key implications for both the occurrence and screening for mitochondrial disorders.

Candy twists as an alternative to the glucola beverage in gestational diabetes mellitus screening.

OBJECTIVE: Screening for gestational diabetes mellitus commonly uses an oral glucose challenge test with a 50-g glucola beverage and subsequent venous puncture. However, up to 30% of pregnant women report significant side-effects, and the beverage is costly. We hypothesized that equivalent glucose loads could be achieved from a popular candy twist (Twizzlers; The Hershey Company, Hershey, PA) and tested it as cost-effective, tolerable alternative with a test of equivalency. STUDY DESIGN: The glucose equivalent of the 50-g glucola was calculated as 10 candy twists. We initially used a triple crossover design in nonpregnant patients whereby each subject served as her own control; this ensured the safety and equivalency of this load before using it among pregnant subjects. We then recruited pregnant women with an abnormal screening at 1 hour (glucose challenge test) in a double crossover design study. Subjects consumed 10 candy twists with a 1-hour venous blood glucose assessment. All subjects subsequently completed the confirmatory 3-hour glucose tolerance test. Sensitivity, specificity, positive predictive values, negative predictive values, false-referral rates, and detection rates were calculated. RESULTS: At ≥130 mg/dL, the sensitivity (100%) was the same for candy twists and glucola. However, the false-referral rate (82% vs 90%), positive predictive value (18% vs 10%), and detection rate (18% vs 10%) were improved for candy twists when compared with the 50-g glucola beverage. CONCLUSION: Our results indicate that strawberry-flavored candy twists are potentially an equally effective screening test, compared with the gold standard glucola beverage but lead to fewer false-positive screens and therefore could be a cost-effective alternative.

Administration of antenatal glucocorticoids and postnatal surfactant ameliorates respiratory distress syndrome-associated neonatal lethality in Erk3(-/-) mouse pups.

BACKGROUND: Respiratory distress syndrome (RDS) persists as a prevalent cause of infant morbidity and mortality. We have previously demonstrated that deletion of Erk3 results in pulmonary immaturity and neonatal lethality. Using RNA sequencing, we identified corticotrophin releasing hormone (CRH) and surfactant protein B (SFTPB) as potential molecular mediators of Erk3-dependent lung maturation. In this study, we characterized the impact of antenatal glucocorticoids and postnatal surfactant on neonatal survival of Erk3 null mice. METHODS: In a double crossover design, we administered dexamethasone (dex) or saline to pregnant dams during the saccular stage of lung development, followed by postnatal surfactant or saline via inhalation intubation. Survival was recorded, and detailed lung histological analysis and staining for CRH and SFTPB protein expression were performed. RESULTS: Without treatment, Erk3 null pups die within 6 h of birth with reduced aerated space, impaired thinning of the alveolar septa, and abundant glycogen stores, as described in human RDS. The administration of dex and surfactant improved RDS-associated lethality of Erk3(-/-) pups and partially restored functional fetal lung maturation by accelerating the downregulation of pulmonary CRH and partially rescuing the production of SFTPB. CONCLUSION: These findings emphasize that Erk3 is integral to terminal differentiation of type II cells, SFTPB production, and fetal pulmonary maturity.

Transcriptome profiling of microRNA by Next-Gen deep sequencing reveals known and novel miRNA species in the lipid fraction of human breast milk.

While breast milk has unique health advantages for infants, the mechanisms by which it regulates the physiology of newborns are incompletely understood. miRNAs have been described as functioning transcellularly, and have been previously isolated in cell-free and exosomal form from bodily liquids (serum, saliva, urine) and tissues, including mammary tissue. We hypothesized that breast milk in general, and milk fat globules in particular, contain significant numbers of known and limited novel miRNA species detectable with massively parallel sequencing. Extracted RNA from lactating mothers before and following short-term treatment with recombinant human growth hormone (rhGH) was smRNA-enriched. smRNA-Seq was performed to generate 124,110,646 36-nt reads. Of these, 31,102,927 (25%) exactly matched known human miRNAs; with relaxing of stringency, 74,716,151 (60%) matched known miRNAs including 308 of the 1018 (29%) mature miRNAs (miRBase 16.0). These miRNAs are predicted to target 9074 genes; the 10 most abundant of these predicted to target 2691 genes with enrichment for transcriptional regulation of metabolic and immune responses. We identified 21 putative novel miRNAs, of which 12 were confirmed in a large validation set that included cohorts of lactating women consuming enriched diets. Of particular interest, we observed that expression of several novel miRNAs were altered by the perturbed maternal diet, notably following a high-fat intake (p<0.05). Our findings suggest that known and novel miRNAs are enriched in breast milk fat globules, and expression of several novel miRNA species is regulated by maternal diet. Based on robust pathway mapping, our data supports the notion that these maternally secreted miRNAs (stable in the milk fat globules) play a regulatory role in the infant and account in part for the health benefits of breast milk. We further speculate that regulation of these miRNA by a high fat maternal diet enables modulation of fetal metabolism to accommodate significant dietary challenges.

Maternal high-fat diet modulates the fetal thyroid axis and thyroid gene expression in a nonhuman primate model.

Thyroid hormone (TH) is an essential regulator of both fetal development and energy homeostasis. Although the association between subclinical hypothyroidism and obesity has been well studied, a causal relationship has yet to be established. Using our well-characterized nonhuman primate model of excess nutrition, we sought to investigate whether maternal high-fat diet (HFD)-induced changes in TH homeostasis may underlie later in life development of metabolic disorders and obesity. Here, we show that in utero exposure to a maternal HFD is associated with alterations of the fetal thyroid axis. At the beginning of the third trimester, fetal free T(4) levels are significantly decreased with HFD exposure compared with those of control diet-exposed offspring. Furthermore, transcription of the deiodinase, iodothyronine (DIO) genes, which help maintain thyroid homeostasis, are significantly (P < 0.05) disrupted in the fetal liver, thyroid, and hypothalamus. Genes involved in TH production are decreased (TRH, TSHR, TG, TPO, and SLC5A5) in hypothalamus and thyroid gland. In experiments designed to investigate the molecular underpinnings of these observations, we observe that the TH nuclear receptors and their downstream regulators are disrupted with maternal HFD exposure. In fetal liver, the expression of TH receptor ß (THRB) is increased 1.9-fold (P = 0.012). Thorough analysis of the THRB promoter reveals a maternal diet-induced alteration in the fetal THRB histone code, alongside differential promoter occupancy of corepressors and coactivators. We speculate that maternal HFD exposure in utero may set the stage for later in life obesity through epigenomic modifications to the histone code, which modulates the fetal thyroid axis.

Maternal tobacco use modestly alters correlated epigenome-wide placental DNA methylation and gene expression.

Several studies linking alterations in differential placental methylation with pregnancy disorders have implicated (de)regulation of the placental epigenome with fetal programming and later-in-life disease. We have previously demonstrated that maternal tobacco use is associated with alterations in promoter methylation of placental CYP1A1 and that these changes are correlated with CYP1A1 gene expression and fetal growth restriction. In this study we sought to expand our analysis of promoter methylation by correlating it to gene expression on a genome-wide scale. Employing side-by-side IlluminaHG-12 gene transcription with Infinium27K methylation arrays, we interrogated correlative changes in placental gene expression and DNA methylation associated with maternal tobacco smoke exposure at an epigenome-wide level and in consideration of signature gene pathways. We observed that the expression of 623 genes and the methylation of 1024 CpG dinucleotides are significantly altered among smokers, with only 38 CpGs showing significant differential methylation (differing by a methylation level of ≥10%). We identified a significant Pearson correlation (≥0.7 or ≤-0.7) between placental transcriptional regulation and differential CpG methylation in only 25 genes among non-smokers but in 438 genes among smokers (18-fold increase, p < 0.0001), with a dominant effect among oxidative stress pathways. Differential methylation at as few as 6 sites was attributed to maternal smoking-mediated birth weight reduction in linear regression models with Bonferroni correction (p < 1.8 × 10(-6)). These studies suggest that a common perinatal exposure (such as maternal smoking) deregulates placental methylation in a CpG site-specific manner that correlates with meaningful alterations in gene expression along signature pathways.

Epigenomics: maternal high-fat diet exposure in utero disrupts peripheral circadian gene expression in nonhuman primates.

The effect of in utero exposure to a maternal high-fat diet on the peripheral circadian system of the fetus is unknown. Using mRNA copy number analysis, we report that the components of the peripheral circadian machinery are transcribed in the nonhuman primate fetal liver in an intact phase-antiphase fashion and that Npas2, a paralog of the Clock transcription factor, serves as the rate-limiting transcript by virtue of its relative low abundance (10- to 1000-fold lower). We show that exposure to a maternal high-fat diet in utero significantly alters the expression of fetal hepatic Npas2 (up to 7.1-fold, P<0.001) compared with that in control diet-exposed animals and is reversible in fetal offspring from obese dams reversed to a control diet (1.3-fold, P>0.05). Although the Npas2 promoter remains largely unmethylated, differential Npas2 promoter occupancy of acetylation of fetal histone H3 at lysine 14 (H3K14ac) occurs in response to maternal high-fat diet exposure compared with control diet-exposed animals. Furthermore, we find that disruption of Npas2 is consistent with high-fat diet exposure in juvenile animals, regardless of in utero diet exposure. In summary, the data suggest that peripheral Npas2 expression is uniquely vulnerable to diet exposure.

In utero tobacco exposure epigenetically modifies placental CYP1A1 expression.

The metabolic pathways used by higher-eukaryotic organisms to deal with potentially carcinogenic xenobiotic compounds from tobacco smoke have been well characterized. Carcinogenic compounds such as polycyclic aromatic hydrocarbons are metabolized sequentially in 2 phases: in phase I, CYP1A1 catalyzes conversion into harmful hydrophilic DNA adducts, whereas in phase II, GSTT1 enables excretion via conjugation into polar electrophiles. In an effort to understand susceptibility to in utero tobacco exposure, we previously characterized known metabolic functional polymorphisms and demonstrated that although deletion of fetal GSTT1 significantly modified birth weight in smokers, no polymorphism fully accounted for fetal growth restriction. Because smoking up-regulates CYP1A1 expression, we hypothesized that nonallelic (epigenetic) dysregulation of placental CYP1A1 expression via alterations in DNA methylation (meCpG) may further modify fetal growth. In the present article, we compared placental expression of multiple CYP family members among gravidae and observed significantly increased CYP1A1 expression among smokers relative to controls (4.4-fold, P < .05). To fully characterize CYP1A1 meCpG status, bisulfite modification and sequencing of the entire proximal 1-kilobase promoter (containing 59 CpG sites) were performed. CpG sites immediately proximal to the 5'-xenobiotic response element transcription factor binding element were significantly hypomethylated among smokers (55.6% vs 45.9% meCpG, P = .027), a finding that uniquely correlated with placental gene expression (r = 0.737, P = .007). Thus, in utero tobacco exposure significantly increases placental CYP1A1 expression in association with differential methylation at a critical xenobiotic response element.

Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production.

Hepatitis C infection causes a state of chronic oxidative stress, which may contribute to fibrosis and carcinogenesis in the liver. Previous studies have shown that expression of the HCV core protein in hepatoma cells depolarized mitochondria and increased reactive oxygen species (ROS) production, but the mechanisms of these effects are unknown. In this study we examined the properties of liver mitochondria from transgenic mice expressing HCV core protein, and from normal liver mitochondria incubated with recombinant core protein. Liver mitochondria from transgenic mice expressing the HCV proteins core, E1 and E2 demonstrated oxidation of the glutathione pool and a decrease in NADPH content. In addition, there was reduced activity of electron transport complex I, and increased ROS production from complex I substrates. There were no abnormalities observed in complex II or complex III function. Incubation of control mitochondria in vitro with recombinant core protein also caused glutathione oxidation, selective complex I inhibition, and increased ROS production. Proteinase K digestion of either transgenic mitochondria or control mitochondria incubated with core protein showed that core protein associates strongly with mitochondria, remains associated with the outer membrane, and is not taken up across the outer membrane. Core protein also increased Ca(2+) uptake into isolated mitochondria. These results suggest that interaction of core protein with mitochondria and subsequent oxidation of the glutathione pool and complex I inhibition may be an important cause of the oxidative stress seen in chronic hepatitis C.

Hepatitis C virus core protein, cytochrome P450 2E1, and alcohol produce combined mitochondrial injury and cytotoxicity in hepatoma cells.

BACKGROUND & AIMS: Alcohol consumption exacerbates liver injury in chronic hepatitis C, and enhanced mitochondrial oxidative stress is one possible mechanism. The aim of this study was to determine whether hepatitis C virus core protein and alcohol-inducible cytochrome P450 2E1 contribute to reactive oxygen species production and cytotoxicity in human hepatoma cells. METHODS: Huh-7 cells expressing core protein, cytochrome P450 2E1, or both were exposed to 0.1 mmol/L tertiary butyl hydroperoxide, tumor necrosis factor alpha, and/or 25 mmol/L ethanol. Cytotoxicity, reactive oxygen species production, glutathione content, and mitochondrial membrane potential were measured. RESULTS: Expression of core/cytochrome P450 2E1 synergistically enhanced cell death induced by either tertiary butyl hydroperoxide or tumor necrosis factor alpha. After tertiary butyl hydroperoxide treatment, total reactive oxygen species production was increased more than 3-fold compared with cells that did not express core and cytochrome P450 2E1. Mitochondrial depolarization and reduced glutathione depletion occurred as well, and cell death was prevented by inhibition of mitochondrial permeability transition or caspase activity. Confocal microscopy showed that the mitochondria themselves were the origin of the reactive oxygen species. In the absence of core/cytochrome P450 2E1 expression, mitochondrial changes and cell death did not occur. Ethanol treatment further decreased mitochondrial reduced glutathione content and exacerbated mitochondrial reactive oxygen species production, depolarization, and cell death. All these effects were prevented by the antioxidant N -acetylcysteine. CONCLUSIONS: Mitochondrial reactive oxygen species production is induced by hepatitis C virus core and cytochrome P450 2E1, resulting in a reduction of mitochondrial antioxidant capacity and sensitivity to oxidants and tumor necrosis factor alpha. Alcohol further depletes mitochondrial reduced glutathione, which exacerbates depolarization and cell death. Sensitization of mitochondria to oxidative insults is thus a potential mechanism for alcohol-related exacerbation of liver injury in chronic hepatitis C.

Plasma appearance and tissue accumulation of non-esterified, free astaxanthin in C57BL/6 mice after oral dosing of a disodium disuccinate diester of astaxanthin (Heptax).

Oral bioavailability of natural and synthetic carotenoids is generally poor in rodents, and this has limited the ability to test these antioxidant compounds in well-defined rodent models of human disease. Various strategies have been employed, with variable success, to increase the percentage of the total oral dose absorbed by the rodent GI tract. In the current study, a novel carotenoid derivative (the disodium disuccinate diester of astaxanthin; Heptax) was administered by oral gavage in a lipophilic emulsion to C57BL/6 mice. Plasma appearance and tissue accumulation of non-esterified, free astaxanthin was studied by HPLC over 72 h after single- and multiple-dose regimens. One-time dosing of Heptax in emulsion at 500 mg/kg resulted in significant appearance of free astaxanthin in plasma (Cmax=0.2 mg/l; 381 nM) and accumulation in solid organs (e.g. liver Cmax=0.9 mg/l; 1735 nM), levels not previously reported after single carotenoid doses in rodents. At each point in the concentration/time curve (AUC), free astaxanthin levels in liver were greater than the corresponding concentration in plasma, suggesting concentrative uptake by the liver. As the ED50 as an antioxidant for non-esterified, free astaxanthin in model systems is approximately 200 nM, the current results suggest that hepatoprotection against oxidative insults may be achieved after a single dose of Heptax in these animals. In humans, where the bioavailability of oral carotenoids ranges from 40 to 60% of the total dose when given in lipophilic vehicle, much smaller oral doses may be utilized for therapeutic benefit in a particular clinical application.

Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein.

BACKGROUND & AIMS: The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood. Indirect evidence suggests that oxidative stress and mitochondrial injury play a role. The aim of this study was to determine if the HCV core protein itself alters mitochondrial function and contributes to oxidative stress. METHODS: HCV core protein was expressed in 3 different cell lines, and reactive oxygen species (ROS) and lipid peroxidation products were measured. RESULTS: Core expression uniformly increased ROS. In 2 inducible expression systems, core protein also increased lipid peroxidation products and induced antioxidant gene expression as well. A mitochondrial electron transport inhibitor prevented the core-induced increase in ROS. A fraction of the expressed core protein localized to the mitochondria and was associated with redistribution of cytochrome c from mitochondrial to cytosolic fractions. Sensitivity to oxidative stress was also seen in HCV transgenic mice in which increased intrahepatic lipid peroxidation products occurred in response to carbon tetrachloride. CONCLUSIONS: Oxidative injury occurs as a direct result of HCV core protein expression both in vitro and in vivo and may involve a direct effect of core protein on mitochondria. These results provide new insight into the pathogenesis of hepatitis C and provide an experimental rationale for investigation of antioxidant therapy.