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2017; 3(9):218–236

RESEARCH ARTICLES

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Chronic Intermittent Hypoxia Causes Lipid Peroxidation and Altered Phase 1 Drug Metabolizing Enzymes in the Neonatal Rat Liver

 

Charles Cai1, Jacob V. Aranda1‒3, Gloria B. Valencia1, Jiliu Xu4, and Kay D. Beharry1‒3.

1Department of Pediatrics, Division of Neonatal-Perinatal Medicine, State University of New York, Downstate Medical Center, Brooklyn, NY 11203, USA; 2Department of Ophthalmology, State University of New York, Downstate Medical Center, Brooklyn, NY 11203, USA; 3SUNY Eye Institute, New York, NY 13202, USA; 4Department of Pediatrics, Division of Gastroenterology, State University of New York, Downstate Medical Center, Brooklyn, NY 11203, USA

Correspondence: kbeharry@downstate.edu (K.D.B.)

Cai C et al. Reactive Oxygen Species 3(9):218–236, 2017; ©2017 Cell Med Press

http://dx.doi.org/10.20455/ros.2017.835

(Received: March 30, 2017; Revised: April 11, 2017; Accepted: April 12, 2017)

ABSTRACT | Critically ill preterm neonates requiring oxygen therapy often experience frequent apneas with intermittent hypoxia (IH). IH-induced oxidative stress causes lipid peroxidation, which targets the liver and contributes to toxic drug reactions. We tested the hypothesis that incremental IH episodes induce oxidative damage in the neonatal liver and alter the expression of genes that regulate drug metabolism. Newborn rats were exposed to increasing IH episodes (12% O2) during hyperoxia (50% O2), or placed in room air (RA) until postnatal day 21 (P21) for recovery from IH (IHR). RA littermates served as controls, and pups exposed to 50% O2 served as hyperoxia controls. Hepatic histopathology, biomarkers of oxidative stress and oxidative DNA damage, antioxidants, and expression of genes that regulate drug metabolism were assessed. Oxidative stress and DNA damage, evidenced by 8-isoprostaglandin F (8-isoPGF) and 8-hydroxy-2′-deoxyguanosine (8-OH-dG), respectively, increased as a function of IH episodes, and was associated with decreased superoxide dismutase (SOD) and increased catalase activities. Pathological changes including cellular swelling, steatosis, necrosis, and focal sinusoid congestion were seen in IH, but not in IHR. Similarly, IH was associated with upregulation of several genes involved in DNA repair, which were downregulated during IHR. Of the genes involved in drug metabolism, aldehyde dehydrogenases (involved in lipid peroxidation) and cytochrome P450 (CYP) genes of the 2C family (involved in oxidative stress) were robustly upregulated both in IH and in IHR. Hepatic oxidative stress and lipid peroxidation occurring in response to chronic IH have implications for preterm infants, and may explain, in part, the pharmacokinetic variations and drug toxicities in this vulnerable population.

KEYWORDS | DNA damage; Hyperoxia; Intermittent hypoxia; Lipid peroxidation; Liver; Oxidative stress; Neonatal rat

ABBREVIATIONS | AOP, apnea of prematurity; CYP, cytochrome P450; ELBW, extremely low birth weight; ELGAN, extremely low gestational age neonate; GPx, glutathione peroxidase; IF, immunofluorescence; IH, intermittent hypoxia; IHR, recovery from IH; 8-isoPGF, 8-isoprostaglandin F; NSAID, non-steroidal anti-inflammatory drug; 8-OH-dG, 8-hydroxy-2′-deoxyguanosine; RA, room air; ROS, reactive oxygen species; SOD, superoxide dismutase


CONTENTS

1. Introduction

2. Material and Methods

2.1. Experimental Design

2.2. IH Cycling

2.3. Oxidative Stress and DNA Damage

2.4. Antioxidant Activities

2.5. Total Cellular Protein Assay

2.6. Hematoxylin and Eosin (H&E) Staining

2.7. Immunofluorescence Staining

2.8. Oxidative Stress and Phase 1 Metabolizing Genes

2.9. Statistical Analyses

3. Results

3.1. Liver 8-isoPGF2α and 8-OH-dG

3.2. Liver Antioxidant Activities

3.3. Liver Histopathology

3.4. Liver Immunofluorescence of CYP Isozymes

3.5. Genes Regulating DNA Damage and Repair

3.6. Phase 1 Metabolizing Genes

4. Discussion


1. INTRODUCTION

Liver dysfunction, clinically manifested by hyperbilirubinemia, affects as many as 50% of extremely low gestational age neonates (ELGANs) and extremely low birthweight (ELBW) infants [1]. Despite multifactorial in etiology, hypoxia/ischemia has been considered one of the major risk factors contributing to hepatic dysfunction in these infants [2]. One of the serious long-term adverse effects associated with early onset liver diseases in ELGANS is an intellectual impairment [3], thus highlighting the need for further mechanistic studies of hypoxia-induced liver injury in neonates. Apnea of prematurity (AOP) with intermittent hypoxia (IH), is a major problem in ELGANS and ELBW infants. The incidence of AOP varies with the degree of prematurity, from 7% at 34–35 weeks of gestational age to 15% at 32–33 weeks, 54% at 30–31 weeks, and nearly 100% at < 28 weeks [4]. Apneas of less than 10 seconds duration can result in a reduction in oxygen saturation of 40% [4]. Studies have shown that ELGANs experience as many as 600 IH episodes per week during the first few weeks of life [5‒8]. These IH events can lead to oxidative stress, which may underpin many of the diseases of the preterm neonate.

Compared to older children and adults, ELGANs are more susceptible to oxidative injury mediated by accumulation of reactive oxygen species (ROS). This is largely due to reduced antioxidant defenses [9‒11]. ELGANs often receive iron supplementation for iron deficiency [12‒15]. Excessive free iron reacts with hydrogen peroxide (H2O2), one of the most abundant ROS, by the Fenton and Haber‒Weis reactions to form the highly toxic hydroxyl radical [16, 17]. The main ROS produced is superoxide anion which is short-lived as it is rapidly degraded to H2O2 and O2 by superoxide dismutase (SOD), the first line of defense [18, 19]. Induction of other antioxidants such as mitochondrial glutathione peroxidase (GPx) and catalase follows to scavenge and detoxify H2O2 in the mitochondria and cytosol. In ELGANS, however, these antioxidant responses are compromised. The H2O2, accumulated from excessive formation and limited clearance, reacts with free iron to form the more reactive from of ROS, hydroxyl radical, which further targets lipids and DNA for oxidative damage [20‒23]. The imbalance between ROS overproduction and antioxidant defenses is of particular significance in the liver due to its high susceptibility to lipid peroxidation [20‒23].

The liver is a prominent organ in neonates. It constitutes approximately 5% of the body weight at birth (compared to 2% in adults) with a large volume (per unit of body weight) of 48 ml/kg [24]. Exposure of the preterm liver to chronic IH can also impair metabolism of many drugs that ELGANs are exposed to including caffeine for AOP, non-steroidal anti-inflammatory drugs (NSAIDs) for closure of a symptomatic patent ductus arteriosus, and antibiotics, among others [23, 25‒27]. ELGANs have shown variable pharmacokinetic profiles of many drugs likely due to immature cytochrome P450 (CYP) system [28‒30]. The combined effects of IH, ROS, and immature drug metabolizing enzymes can contribute to severe hepatotoxicity. Studies show that H2O2 and other ROS diminish hepatic expression and activity of CYP enzymes [31‒36].

Based upon these reports, we tested the hypothesis that increasing episodes of IH followed by IHR cause substantial ROS damage to the liver and alter the expression of phase 1 drug metabolizing enzymes. To prove this hypothesis, we exposed neonatal rats to increasing episodes of neonatal IH and examined the responses of hepatic biomarkers of oxidative stress, oxidative DNA damage, antioxidants, and CYP gene expression.

2. MATERIAL AND METHODS

All experiments were approved by the State University of New York, Downstate Medical Center Institutional Animal Care and Use Committee, Brooklyn, NY, USA. Animals were treated humanely, according to the guidelines outlined by the United States Department of Agriculture and the Guide for the Care and Use of Laboratory Animals.

2.1. Experimental Design

Certified infection-free, timed-pregnant Sprague Dawley rats were purchased from Charles River Laboratories (Wilmington, MA, USA) at 17 days of gestation. The animals were housed in an animal facility with a 12-hour-day/12-hour-night cycle and provided standard laboratory diet and water ad libitum until delivery. Within 2‒3 hours of birth, newborn pups delivered on the same day were pooled and randomly assigned to expanded litters of 18 pups/litter (9 males and 9 females). Gender was determined by the anogenital distance. The expanded litter size was used to simulate relative postnatal malnutrition of critically ill ELGANs. Each pup was weighed and measured for linear growth (crown to rump length in centimeters). A total of 31 groups of 18 rat pups (9 males and 9 females) were studied according to the experimental design previously published [34].

The groups are described as follows: (1) groups 1 to 6 were exposed to 2, 4, 6, 8, 10, or 12 IH cycling episodes from P0 to P7; (2) groups 7 to 12 were exposed to 2, 4, 6, 8, 10, or 12 IH cycling episodes from P0 to P14; (3) groups 13 to 18 were exposed to 2, 4, 6, 8, 10, or 12 IH cycling episodes from P0 to P7, followed by re-oxygenation in room air (RA) for 14 days from P7 to P21; (4) groups 18 to 24 were exposed to 2, 4, 6, 8, 10, or 12 IH cycling episodes from P0 to P14, followed by re-oxygenation in RA for 7 days from P14 to P21; (5) groups 25 to 28 were exposed to 50% O2 only for 7 days, 14 days, 7 days with 14 days of re-oxygenation in RA, or 14 days with 7 days of re-oxygenation in RA. These served as O2 controls, or zero IH; and (6) groups 29‒31 were littermates raised in RA from birth to P7, P14, or P21 with all conditions identical except for atmospheric oxygen and served as RA controls.

2.2. IH Cycling

The IH cycles consisted of hyperoxia (50% O2)/hypoxia (12% O2) in stepwise increments of brief (1 min), hypoxia (12%) clusters (3 clusters) during 50% O2 for a total of 2, 4, 6, 8, 10, or 12 episodes/day. This clustering design has been shown to produce severe oxidative stress in neonatal rats [35‒40]. There were two IH groups corresponding to 7-day (P0‒P7), and 14-day (P0‒P14). To determine the immediate effects, animals were euthanized on P7 or P14. To determine the effects of IHR, all animals were euthanized on P21 after either 14 days of recovery in RA following 7-day IH (P7‒P21), or 7 days of recovery in RA following 14-day IH (P14‒P21).

2.3. Oxidative Stress and DNA Damage

8-Isoprostane (8-isoPGF) is commonly studied and abundantly generated in vivo during oxidative stress and lipid peroxidation. To establish ocular oxidative stress and DNA damage, levels of 8-isoPGF and 8-hydroxy-2′-deoxyguanosine (8-OH-dG), respectively, were determined in the liver homogenates using commercially-available enzyme immunoassay kits (Assay Designs, Ann Arbor, MI, USA), according to the manufacturer’s protocol. Levels in the homogenates were standardized using total cellular protein levels according to the Bradford method (Bio-Rad, Hercules, CA, USA).

2.4. Antioxidant Activities

Superoxide dismutase (SOD), catalase activities were determined in the homogenates using commercially-available assay kits (Cayman Chemicals, Ann Arbor, MI, USA) according to the manufacturer’s protocol. The SOD and catalase activities were determined spectrophotometrically at 450 nm and 540 nm, respectively. Levels were standardized using total cellular protein levels.

2.5. Total Cellular Protein Assay

On the day of the assay, liver homogenates were assayed for total protein levels using the dye-binding Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard.

2.6. Haemotoxylin and Eosin (H & E) Staining

Liver biopsies were fixed in 10% neutral buffered formalin (NBF) and sent to Histowiz (Brooklyn, NY, USA) for processing, sectioning, and H&E staining. Images were captured at 40× magnification using an Olympus BX53 microscope, DP72 digital camera, and CellSens imaging software (Olympus, Center Valley, PA, USA), attached to a Dell Precision T3500 computer (Dell, Round Rock, TX, USA). Unstained sections were used for immunofluorescence staining of CYP1A2 and CYP2D6.

2.7. Immunofluorescence Staining

 Unstained sections were de-paraffinized and treated with xylenes and ethanol. The slides were placed in 10 mM sodium citrate buffer, pH 6.0 and heated at 95‒100oC for 20 minutes to unmask the antigens. Following several washes, Immunofluorescence staining (IF) staining was conducted using primary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) and Alexa Fluor fluorescent secondary antibodies (Life Technologies, Grand Island, NY, USA). All IF protocols were conducted according to the manufacturer’s recommendations. IF sections were imaged at 40× magnification using an Olympus BX53 microscope, DP72 digital camera, and CellSens imaging software (Olympus, Center Valley, PA, USA), attached to a Dell Precision T3500 computer (Dell, Round Rock, TX, USA).

2.8. Oxidative Stress and Phase 1 Metabolizing Genes

Total RNA was extracted as previously described [39‒41]. Real-time polymerase chain reaction (PCR) arrays were carried out in duplicate using the rat oxidative stress and phase 1 metabolizing gene arrays (Qiagen, Germantown, MD, USA) with a Bio-Rad IQ5 real-time instrument (Bio-Rad, Hercules, CA, USA) according to the manufacturers’ protocols.

2.9. Statistical Analyses

Data were analyzed in two ways: (1) comparison of 50% O2 and IH groups to RA; and (2) comparison IH to IHR. A test for normality was conducted using the Bartlett’s test, prior to all statistical analyses. Normally distributed data was analyzed using two-way analysis of variance with Bonferroni post-hoc tests. Non-normally distributed data was analyzed using Kruskall Wallis test with Dunn’s multiple comparison test. Data are presented as mean ± SEM (n = 8) and a p value of < 0.05 was considered as statistically significant, using SPSS version 16.0 (SPSS, Chicago, IL, USA) and graphs were prepared using GraphPad Prizm (GraphPad, San Diego, CA, USA).

3. RESULTS

3.1. Liver 8-isoPGF and 8-OH-dG

Figure 1 represents the effects of increasing IH episodes (white bar) and subsequent IHR (black bar) on 8-isoPGF and 8-OH-dG in neonatal rat liver homogenates. In the 7-day IH groups, the levels of 8-isoPGF were substantially higher compared to the IHR groups. Exposure to 10 and 12 IH episodes per day evoked a profound increase in liver 8-isoPGF levels. IHR for 14 days resulted in consistently lower 8-isoPGF levels with a moderate increase in the hyperoxia group compared to RA (Figure 1A). Exposure to 14-day IH resulted in higher 8-isoPGF with 8‒12 IH episodes, as did exposure to hyperoxia alone. While IHR for 7 days following either hyperoxia or IH decreased 8-isoPGFlevels, IHR following 6 IH episodes caused an increase (Figure 1B). In the 7-day IH groups, liver 8-OH-dG levels decreased with hyperoxia only and progressively increased with the number of IH episodes to peak with 10 IH episodes. In the 14-day IHR groups the levels declined in the hyperoxia groups and groups exposed to 4, 6, and 10 IH groups (Figure 1C). In the 14-day IH groups, the effects on 8-OH-dG appeared to be also lower with hyperoxia, but to a lesser degree, and peaked with 10 IH episodes. IHR was lower in the 10 IH group only, compared to IH (Figure 1D).

FIGURE 1. Effects of incremental IH episodes on 8-isoPGF (A, B) and 8-OH-dG (C, D) in the liver of neonatal rats at P7 (7D-IH), P14 (14D-IH), or P21-IHR. Animals recovering from 7D-IH were placed in RA from P7‒P21 and animals recovering from 14D-IH were placed in RA from P14‒P21. Animals randomized to IH were exposed to brief clustered IH episodes during 50% O2. Animals randomized to hyperoxia were exposed to 50% O2 only. RA controls remained in atmospheric O2. Data are presented as mean ± SEM (n=8 samples/group).

3.2. Liver Antioxidant Activities

Figure 2 represents the effects of increasing IH episodes (white bar) and subsequent IHR (black bar) on SOD (A and B) and catalase (C and D) activities in the liver homogenates. In the 7-day IH group, liver SOD activities were highest with 2 IH episodes per day and progressively declined with 4‒12 IH episodes per day. IHR following 7-day IH exposure  followed a similar pattern and were lower than IH levels with 8, 10, and 12 IH episodes (Figure 2A). Exposure to chronic IH (14-day) episodes displayed a similar pattern to 7-day IH exposure, but with a higher peak production with 2 IH episodes, although lower levels were found with recovery compared to IH (Figure 2B). In the 7-day IH group, liver catalase levels declined with hyperoxia and with 2‒6, and 12 IH episodes.  Exposure to 10 IH episodes increased catalase levels. During IHR, the levels were higher with 2 and 4 IH episodes, and lower with 6‒12 IH episodes.  Catalase levels did not change during recovery from hyperoxia (Figure 2C). Exposure to long-term IH caused significant increases in catalase with 8 and 10 episodes. Both hyperoxia and 12 IH episodes caused lower catalase levels. During IHR, catalase levels remained consistently lower in the hyperoxia group and in the 8‒10 IH groups (Figure 2D).

FIGURE 2. Effects of incremental IH episodes on SOD (A, B) and catalase (C, D) activities in the liver of neonatal rats at P7 (7D-IH), P14 (14D-IH), or P21-IHR. Animals recovering from 7D-IH were placed in RA from P7‒P21 and animals recovering from 14D-IH were placed in RA from P14‒P21. Animals randomized to IH were exposed to brief clustered IH episodes during 50% O2. Animals randomized to hyperoxia were exposed to 50% O2 only. RA controls remained in atmospheric O2. Data are presented as mean ± SEM (n=8 samples/group).

3.3. Liver Histopathology

Figure 3 shows normal histology of liver tissue from RA animals at P7 (A), P14 (B), and P21 (C and D). Figure 4 shows liver sections from animals exposed to 7 days of 2 (A), 4 (B), 6 (C), 8 (D), 10 (E), and 12 (F) IH episodes per day.  Increasing episodes of IH caused cellular swelling, steatosis, necrosis and focal sinusoid congestion starting as early as 2 IH episodes per day. More severe apoptosis occurred with 6 IH episodes per day. No major damage was seen with 8 and 12 IH episodes. Figure 5 shows liver sections from animals exposed to 14 days of 2 (A), 4 (B), 6 (C), 8 (D), 10 (E), and 12 (F) IH episodes per day. While the damage was less severe than that seen after 7 days of IH exposure, there was evidence of necrosis and steatosis in the groups exposed to 6‒10 IH episodes. Exposure to 12 IH episodes showed evidence of blood sinusoid dilation and hemorrhage. Figure 6 shows liver sections from animals exposed to 7 days of 2 (A), 4 (B), 6 (C), 8 (D), 10 (E), and 12 (F) IH episodes per day with 14 days of IHR. IHR in all groups showed no major evidence of necrosis, but cytoplasmic vacuolization, pervasive steatosis, blood sinusoid dilation and hemorrhage occurred particularly in the 12 IH/IHR group. Figure 7 shows liver sections from animals exposed to 14 days of 2 (A), 4 (B), 6 (C), 8 (D), 10 (E), and 12 (F) IH episodes per day with 7 days of IHR. IHR in all groups showed no major evidence of necrosis, but similar to the 14-day IHR groups, there was pervasive steatosis, blood sinusoid dilation, hemorrhage, and cytoplasmic vacuolization.

FIGURE 3. Representative H & E stain of liver sections from RA controls at P7 (A), P14 (B) and P21 (C and D), and representative immunofluorescence stain of CYP1A2 (E) and CYP2D6 (F) from RA controls at P21. Control liver sections show normal architecture and robust immunostaining in the hepatocytes. Images are 40× magnification. Scare bar represents 20 µm.

FIGURE 4. Representative H & E stain of liver sections from 7D-IH groups exposed to 2 (A), 4 (B), 6 (C), 8 (D), 10 (E), and 12 (F) IH episodes per day. Increasing episodes of IH caused cellular swelling, steatosis, necrosis and focal sinusoid congestion starting as early as 2 IH episodes per day (arrows). More severe apoptosis occurred with 6 IH episodes per day. No major damage was seen with 8 and 12 IH episodes. Images are 40× magnification. Scare bar represents 20 µm.

FIGURE 5. Representative H & E stain of liver sections from 14D-IH groups exposed to 2 (A), 4 (B), 6 (C), 8 (D), 10 (E), and 12 (F) IH episodes per day. Images show evidence of necrosis and steatosis in the groups exposed to 6-10 IH episodes (arrows). Exposure to 12 IH episodes showed evidence of blood sinusoid dilation and hemorrhage (arrows). Images are 40× magnification. Scare bar represents 20 µm.

FIGURE 6. Representative H & E stain of liver sections from 21-day old rats exposed to 7 days of IH and 14 days of IHR, and exposed to 2 (A), 4 (B), 6 (C), 8 (D), 10 (E), and 12 (F) IH episodes per day.  IHR in all groups showed no major evidence of necrosis, but there was and cytoplasmic vacuolization, pervasive steatosis, blood sinusoid dilation and hemorrhage, particularly in the 12 IH/IHR group (arrows). Images are 40× magnification. Scare bar represents 20 µm.

FIGURE 7. Representative H & E stain of liver sections from 21-day old rats exposed to 14 days of IH and 7 days of IHR, and exposed to 2 (A), 4 (B), 6 (C), 8 (D), 10 (E), and 12 (F) IH episodes per day. IHR in all groups showed no major evidence of necrosis, but similar to the 14-day IHR groups, there was pervasive steatosis, blood sinusoid dilation, hemorrhage, and cytoplasmic vacuolization (arrows). Images are 40× magnification. Scare bar represents 20 µm.

3.4. Liver Immunofluorescence of CYP Isozymes

Figure 3E and 3D shows expression of CYP1A2 and CYP2D6, respectively in hepatocytes of livers from control rats at P21. Figures 8 and 9 shows significantly reduced intensity in all groups exposed to 14 days of IH with 7 days of IHR, although CYP1A2 was moderately expressed in the group exposed to 2 IH episodes per day (Figure 8A).

FIGURE 8. Representative immunofluorescence stain CYP1A2 in liver sections from 21-day old rats exposed to 14 days of IH and 7 days of IHR, and exposed to 2 (A), 4 (B), 6 (C), 8 (D), 10 (E), and 12 (F) IH episodes per day. CYP1A2 was significantly decreased in all areas of the liver. Images are 40× magnification. Scare bar represents 20 µm (note: refer to Figure 3E for control).

FIGURE 9. Representative immunofluorescence stain CYP2D6 in liver sections from 21-day old rats exposed to 14 days of IH and 7 days of IHR, and exposed to 2 (A), 4 (B), 6 (C), 8 (D), 10 (E), and 12 (F) IH episodes per day. CYP2D6 was significantly decreased in all areas of the liver. Images are 40× magnification. Scare bar represents 20 µm (note: refer to Figure 3D for control).

3.5. Genes Regulating DNA Damage and Repair

Expression of genes regulating DNA damage and repair after 14 days of IH is presented in Table 1, and the expression of the same genes after 14 days of IH and 7 days IHR are presented in Table 2. Data are expressed as fold changes as compared to P14 or P21 RA controls. A total of 18 genes are selected from a panel of 84 genes involved in the regulation of DNA damage, DNA repair, apoptosis, and cell cycle. Samples from 4‒10 IH episodes for 14 days and the corresponding IHR at 21 days are presented to correlate with data from Figures 1 and 2. Concurrent with the results shown in Figures 1 and 2, exposure to IH resulted in higher expression of genes compared to IHR. During IHR, most genes remained downregulated.

TABLE 1. DNA damage genes in the rat liver after 14 days of IH
Genes of Interest 4 IH Cycles/Day 6 IH Cycles/Day 8 IH Cycles/Day 10 IH Cycles/Day
Apex 11.9 3.6 7.8 14
Brca1 7 4.2 9.2 14.6
Ercc2 5.3 2.3 39.2 4.7
Hprt1 6.5 22 16.4 18.3
Mare 24.3 79.5 5.7 39.6
Mbd4 7.4 8.5 7.5 10
Mgmt 2.8 9.3 1.4 6.7
Mif 4.7 29.0 2 12.3
Msh2 10 29 4.3 15.5
Msh3 8.2 14.6 15.8 15.7
P53 4.7 3.7 4.1 8.6
Parp1 2.8 3.5 3.8 7
Rad1 5.1 10.1 2.2 14.5
Rad21 3.2 3.9 1.7 10.1
Rad23a 4.6 8.9 2.1 6.8
Rad51 3.1 11.3 12 7
Rad51c 2.6 9.2 2.1 5.5
Ube2a 3.4 11.7 3.1 9.4
Note: Data are fold change from P14 RA control. All data were corrected using 5 different housekeeping genes. Genes are selected from a profile of 84 genes. Genes of interest are: Apex (APEX nuclease multifunctional DNA repair enzyme 1); Brca1, (breast cancer 1); Ercc2 (excision repair cross-complementing rodent repair); Hprt1 (hypopxanthine phosphoribosyltransferase 1); Mare (alpha globin regulatory element containing gene); Mbd4 (methyl-CpG binding domain protein 4); Mgmt (O-6-methylguanine-DNA methyltransferase); Mif (macrophage migration inhibitory factor); Msh2 (MutS homolog 2); Msh3 ((MutS homolog 3); P53 (tumor suppressor protein 53); Parp 1 (poly ADP-ribose polymerase 1); Rad 1, 21, 23a, 51, 51c (RAD1, 21, 23a, 51, 51c homolog); Ube2a (ubiquitin-conjugating enzyme E2A).

 

TABLE 2. DNA damage genes in the 21-day old rat liver after 14 days of IH and 7 days of IHR
Genes of Interest 4 IH Cycles/Day 6 IH Cycles/Day 8 IH Cycles/Day 10 IH Cycles/Day
Apex ‒1.8 1.5 2.6 1.5
Brca1 1.2 ‒3.9 1.9 1.5
Ercc2 ‒2.9 ‒1.3 ‒1.3 ‒3.9
Hprt1 1.9 1.3 13.1 5.9
Mare ‒1.8 ‒1.5 2.5 1.3
Mbd4 ‒1.2 ‒1.1 1.3 1.1
Mgmt ‒2.6 ‒4.9 2.1 1.3
Mif ‒1.4 ‒3 2 1.1
Msh2 ‒1.8 1.1 1.5 1.2
Msh3 ‒1.6 ‒1.5 2.4 1.1
P53 ‒1.1 ‒1.4 2.3 2.2
Parp1 ‒1.6 ‒1.5 ‒1.2 ‒1.1
Rad1 ‒1.9 ‒1.4 2.6 1
Rad21 ‒1.7 3.5 5.3 1.9
Rad23a ‒1.8 ‒1.7 ‒1 ‒1
Rad51 ‒1.6 ‒2.3 2 1.8
Rad51c ‒2 1 2.1 ‒1
Ube2a ‒1.7 1.0 3.9 2.1
Note: Data are fold change from P21 RA control. Genes of interest are as described in Table 1. Downregulated genes are presented with a minus sign.

3.6. Phase 1 Metabolizing Genes

Expression of genes encoding proteins that are involved in phase 1 drug metabolism following 14 days of IH and 7 days of IHR are presented in Tables 3 and 4, respectively. A total of 22 genes are selected from a panel of 84 genes. Aldehyde dehydrogenase genes, most notably Aldh6 (139-fold upregulation), were several fold upregulated with 10 IH episodes after 14 days of exposure, as was cycloxygenase-2 (COX-2), the inducible form of COX associated with inflammation. In contrast, most CYP genes, whose encoded proteins are flavin-containing monooxygenases, responsible for catalyzing the oxidation of many drugs, were downregulated with increasing IH episodes (Table 3). Compared to P14, expression of genes involved in phase 1 drug metabolism was substantially lower during IHR, except for Aldh2 (59-fold), Aldh6 (> 230-fold), inducible COX-2 (24-fold), the retinoid inducible Cyp2c22 (> 86-fold), Cyp2e1 (> 60-fold), and Fm05 (35-fold) which were robustly upregulated (Table 4).

TABLE 3. Phase 1 drug metabolizing genes in the rat liver after 14 days of IH
Genes of Interest 4 IH Cycles/Day 6 IH Cycles/Day 8 IH Cycles/Day 10 IH Cycles/Day
Aldh1a1 22.2 9.5 4.0 28.6
Aldh2 32.3 83.3 3.0 41.8
Aldh4 41.7 45.3 10.3 60.3*
Aldh5a1 19.9 32.6 4.2 23.0
Aldh6 21.8 64.8 8.9 139.3*
Aldh8a1 44.8 34.0 7.5 22.2
COX-1 15.9 19.7 2.5 34.6*
COX-2 3.2 2.0 1.2 14.3*
Cyp1a1 4.7 1.8 1.5 12.5
Cyp1a2 12.8 9.1 2.2 13.6
Cyp2a3 10.0 21.2 5.3 28.1
Cyp2b2 33.2 60.0 2.0 38.0
Cyp2c11 22.4 43.3 4.1 16.8
Cyp2c22 20.5 77.1 ‒2.1 34.0
Cyp2c23 12.1 34.5 4.2 15.3
Cyp2d2 7.1 2.6 0 1.9
Cyp2d4 9.1 19.9 1.8 17.6
Cyp2e1 35.7 39.0 11.8 47.3*
Cyp2f4 24.1 11.2 3.4 16.4
Cyp8b1 82.6 65.4 6.1 9.5
Fmo1 77.7 26.2 13.6 19.6*
Fmo5 33.9 34.9 3.6 62.1*
Xdh 2.7 3.8 ‒1.8 8.7
Note: Data are fold change from P14 RA control. Genes of interest are: aldh1a1 (aldehyde dehydrogenase 1 family, member A1); aldh2 (aldehyde dehydrogenase 2 family, mitochondrial); aldh4 (alcohol dehydrogenase 4 (class II), pi polypeptide); aldh5a1 (aldehyde dehydrogenase 5 family, member A1); aldh6 (alcohol dehydrogenase 6, class V); aldh8a1 (aldehyde dehydrogenase 8 family, member A1); COX-1 (cyclooxygenase 1); COX-2 (cyclooxygenase 2); Cyp1a1, 1a2 (cytochrome P450, family 1, subfamily a, polypeptide 1, 2); Cyp2a3 (cytochrome P450, family 2, subfamily a, polypeptide 3); Cyp2b2 (cytochrome P450, family 2, subfamily b, polypeptide 2); Cyp2c11, 22, 23 (cytochrome P450, family 2, subfamily c, polypeptide 11, 22, 23); Cyp2d2, 4 (cytochrome P450, family 2, subfamily d, polypeptide 2, 4); Cyp2e1 (cytochrome P450, family 2, subfamily e, polypeptide 1); Cyp2f4 (cytochrome P450, family 2, subfamily f, polypeptide 4); Cyp8b1 (cytochrome P450, family 8, subfamily b, polypeptide 1); Fmol1, 5 (flavin containing monooxygenase 1, 5); Xdh (xanthine dehydrogenase). Downregulated genes are presented with a minus sign. *genes associated with lipid peroxidation and inflammation.

 

TABLE 4. Phase 1 drug metabolizing genes in the 21-day old rat liver after 14 days of IH and 7 days of IHR
Genes of Interest 4 IH Cycles/Day 6 IH Cycles/Day 8 IH Cycles/Day 10 IH Cycles/Day
Aldh1a1 5.4 10.6 7.2 14.2
Aldh2 7.4 7.7 5.3 59.1*
Aldh4 6.8 1.9 4.5 20.2*
Aldh5a1 6.2 3.5 6.8 19.1*
Aldh6 47.3 21.7 7.5 230.7*
Aldh8a1 4.4 4.9 4.5 9.7
COX-1 1.8 4.7 2.7 24.3
COX-2 2.1 15.0 21.6 28.7*
Cyp1a1 18.0 3.3 4.1 4.8
Cyp1a2 5.5 ‒1.3 1.6 4.5
Cyp2a3 10.7 ‒1.0 6.1 8.1
Cyp2b2 4.1 1.3 4.0 7.9
Cyp2c22 15.5 11.5 8.9 86.8*
Cyp2c23 13.1 1.2 5.2 21.8
Cyp2d2 ‒1.2 ‒1.4 ‒1.9 ‒2.1
Cyp2d4 4.7 1.0 2.4 15.4
Cyp2e1 18.0 1.8 2.4 60.6*
Cyp2f4 6.3 11.1 4.1 15.0
Cyp8b1 9.4 1.9 5.0 2.8
Fmo1 13.1 6.6 3.9 12.9
Fmo5 8.0 5.5 4.3 35.4*
Xdh 2.7 1.5 4.3 10.1
Note: Data are fold change from P21 RA control animals. Genes of interest are as described in Table 3. Downregulated genes are presented with a minus sign. *genes associated with lipid peroxidation and inflammation.

4. DISCUSSION

The results of this study show that IH and subsequent IHR, inflict detrimental effects to the neonatal liver via oxidative stress and DNA damage. The importance of these findings relates specifically to the significant pharmacologic variations that exist among preterm neonates, as well as the substantial variation in pharmacokinetic profiles of drugs which is most likely due to immature drug metabolizing enzymes [28‒30]. Since drug therapies in ELGANs are largely based on data from older children and adults, one important factor that has not been considered but may contribute to toxicokinetics and toxicodynamics in this vulnerable population, is the effect of IH on drug metabolizing enzymes.

Critically ill preterm neonates are exposed to a multitude of off-label drugs including methylxanthines, NSAIDs, antibiotics, narcotics, etc. for various morbidities. The combination of polypharmacy, recurring IH/IHR episodes, and immature and/or deficient drug metabolizing systems contribute to toxic drug reaction. Drug metabolizing enzymes are divided into phase 1 and phase 2 enzymes. Phase 1 enzymes are involved primarily with oxidation, reduction and hydrolysis processes, while phase 2 enzymes conjugate drug molecules to allow excretion [41]. The most important group of enzymes involved in phase 1 metabolism are CYP enzymes. Deficient or immature activity of CYP enzymes can be responsible for the extreme toxicity seen in preterm neonates [42‒44]. At birth, total neonatal hepatic CYP content is approximately 30% of adult levels [45], and increases at variable rates during the perinatal period [44‒47]. CYP3A4 is responsible for the metabolism of a vast majority of currently marketed drugs. It is the most abundantly expressed CYP enzyme in the liver, accounting for 30‒40% of total CYP enzyme content in the organ, and is functionally immature at birth reaching 50% of adult levels by 6‒12 months of age [48]. In this study, we found no significant effects of IH on CYP3A4. This is likely due to the immature function of this CYP isozyme. Other metabolic deficiencies include decreased hydroxylating and esterase activities [49] and glucuronidation mechanisms [50].

The hepatic glucuronyltransferase system in the neonatal rat at birth is not fully developed. At day 14, it is functional at or close to maximal efficiency, and at 21 days postnatal age is functional at or close to adult capacity [51]. Using validated biomarkers for oxidative stress and DNA damage, 8-isoPGF and 8-OH-dG, respectively [52, 53], we showed that IH causes oxidative stress and DNA damage to the liver. Oxidative stress was significantly more severe in the groups experiencing10 and 12 IH episodes per day despite the shorter hyperoxic events between IH episodes compared to the other groups. This clearly demonstrates that hypoxia can exert more oxidative damaging effects on the liver than hyperoxia. RA recovery is unable to appreciably mitigate the effects. Oxidative stress and propagation of ROS target the liver due to its high lipid content, resulting in lipid peroxidation [20‒23]. Studies show that H2O2, as well as other ROS diminishes the expression, content, and activity of CYP isozymes [31]. In addition, CYP isozymes undergo uncoupled catalytic turnover, resulting in the formation of superoxide, H2O2, and singlet oxygen [32, 54, 55]. Thus, while CYP enzymes play important roles in maintaining therapeutic level of a drug in the body, they continuously produce ROS as an inevitable result of oxidative drug metabolism. Adverse drug effects are also attributable to CYP ROS generation. ROS disrupted CYP1A2 and contributed to acetaminophen liver toxicity [21] and theophylline metabolism [27].

Superoxide dismutase (SOD) is the first line of defense against superoxide anion, and is the primary ROS detoxifying enzyme [56]. Three forms of SOD exist in the cell, namely, copper-and zinc containing SOD (CuZnSOD, or SOD-1), manganese-containing SOD (MnSOD, or SOD-2); and extracellular SOD (ECSOD, or SOD-3) [57]. All forms of SOD catalyze the dismutation of superoxide anion into H2O2 and O2. The levels of SOD fluctuate in response to the amount of superoxide anion. The cell uses multiple enzyme systems to catalyze the decomposition of H2O2 into water and O2. One of the systems involves glutathione peroxidase (GPx), two forms of which have been identified in mitochondria [57, 58]. Catalase is another important enzyme used by cells to decompose H2O2 [59, 60]. Catalase is found primarily in peroxisomes, but can also be present in the cytoplasm.  In our study, it was interesting to note that as SOD levels decreased with increasing IH, the levels of catalase increased. These findings clearly demonstrate that accumulation of H2O2 with higher IH episodes may be the predominant ROS, as previously demonstrated by our group [34], which may be responsible for the lipid peroxidation, reduction in the expression of phase 1 metabolizing enzymes, and damage to the liver [61].

Our study found that the younger newborn rats at 7 days had more tissue damage compared to the older rats at P14 and P21, suggesting tissue repair and regeneration in the older rats. This is evident by the increased expression of genes that regulate DNA damage and repair. Over 90% of all ELGANs are treated with caffeine for AOP. Caffeine is metabolized in the liver primarily by CYP1A2, which accounts for 95% of caffeine clearance. CYP1A2 is developmentally delayed in human fetal liver and is the last major CYP isoform to develop during the first postnatal trimester [62]. Some 25% of all clinically used drugs are metabolized by CYP2D6 [63], several of which are commonly used in newborns and neonates [46]. However, like CYP1A2, CYP2D6 is increased after birth [64]. For these reasons, we examined the expression of CYP1A2 and CYP2D6 in the P21 rats only and found that exposure to IH, even at low numbers of episodes, suppresses the expression of both CYP1A2 and CYP2D6 in the liver. The consequences of this can have major implications in the preterm infant such as therapeutic failure and toxicity.

One of the most significant findings was the robust expression of aldehyde dehydrogenases (Aldh), particularly Aldh6 after 14 days of 10 IH episodes per day, and to a greater extent at P21, after 7 days of IHR. Aldehydes are generated during a series of physiological processes with more than 200 aldehyde species arising from lipid peroxidation [64]. Aldehydes are detoxified by Aldh which protects the cells against lipid peroxidation. The upregulation of Aldh during increased IH episodes may represent a compensatory response to counteract increasing lipid peroxidation in the liver. To our knowledge, this is the first study to demonstrate this phenomenon, and these data suggest that Aldh may represent a novel therapeutic target against lipid peroxidation. Another interesting finding was the increased expression of Cyp2c22 and Cyp2e1 after chronic IH exposure. The liver contains abundant members of CYP2C family metabolizing drugs such as paclitaxel (CYP2C8) and NSAIDs (CYP2C9). CYP2C22, sharing 80% similarity with CYP2C9, is predominantly expressed in the liver, and is involved in retinoic acid metabolism [64]. Interestingly, retinoic acid is also involved in oxidative stress [65]. CYP2E1 is also intimately involved in oxidative stress in the liver [66], and development of steatosis and apoptosis [67]. Taken together, these findings clearly indicate that increasing IH is closely associated with hepatic lipid peroxidation, which may not be ameliorated but further enhanced by IHR.

In conclusion, the liver appears to be tolerant of short-term, but not prolonged IH exposure. Less IH events produced more SOD possibly in response to increased superoxide anion, while more IH events produced more catalase which may indicate accumulation of H2O2. Long-term IH events produced more severe oxidative stress and DNA damage. This may have implications for preterm infants who have immature drug metabolizing systems, experience frequent oxygen desaturations and apneas during oxygen therapy, and exposed to numerous off-label drugs. Finally, suppression of key genes responsible for metabolism of drugs commonly used in the preterm neonate in the setting of IH may be orchestrated by H2O2, a master contributor to lipid peroxidation, and possibly, other toxic reactions.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health‒Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda MD, USA (Grant No. 1U54HD071594). Authors declare no conflicts of interest related to the work reported in this paper.

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