Chronic exposure to hypoxia during pregnancy generates a stressed intrauterine environment that may lead to fetal organ damage. The objectives of the study are (1) to quantify the effect of chronic hypoxia in the generation of oxidative stress in fetal guinea pig liver and (2) to test the protective effect of antioxidant treatment in hypoxic fetal liver injury. Pregnant guinea pigs were exposed to either normoxia (NMX) or 10.5% O 2 (HPX, 14 days) prior to term (65 days) and orally administered N-acetylcysteine ([NAC] 10 days). Near-term anesthetized fetuses were excised and livers examined by histology and assayed for malondialdehyde (MDA) and DNA fragmentation.

Chronic HPX increased erythroid precursors, MDA (NMX vs HPX; 1.26 ± 0.07 vs 1.78 ± 0.07 nmol/mg protein; P .001, mean ± standard error of the mean [SEM]) and DNA fragmentation levels in fetal livers (0.069 ± 0.01 vs 0.11 ± 0.005 OD/mg protein; P .01).Difference between hypoxia and anoxia N-acetylcysteine inhibited erythroid aggregation and reduced ( P .05) both MDA and DNA fragmentation of fetal HPX livers. Thus, chronic intrauterine hypoxia generates cell and nuclear damage in the fetal guinea pig liver. Maternal NAC inhibited the adverse effects of fetal liver damage suggestive of oxidative stress. The suppressive effect of maternal NAC may implicate the protective role of antioxidants in the prevention of liver injury in the hypoxic fetus.

Introduction

Chronic hypoxia in utero is a major cause of fetal growth restriction and fetal and neonatal morbidity and mortality. 1 – 3 yet, the impact of chronic exposure to hypoxia on fetal organ function has had limited study. The hypoxic fetus redistributes its cardiac output to favor both the heart and the brain at the expense of the fetal liver. 4 – 6 thus, the fetal liver is vulnerable to damage under conditions of chronic fetal hypoxia.Difference between hypoxia and anoxia

The fetal liver plays a primary role in hemopoiesis during the fetal period and transitions to a more metabolic role near term or after birth depending on the animal species. The fetal liver consists of hemopoietic cells, hepatocytes, as well as, sinusoidal and bile ductal cells during its development. 7, 8 during the fetal period, erythropoietin is produced in hepatocytes and fibroblast-like ito cells as evidenced by its gene expression in the liver. 9, 10 with the gestational proliferation of hepatocytes near term, hepatocytes begin to play an important role in carbohydrate 11 and lipid 12 metabolism, as well as, synthesis and secretion of liver proteins 13 associated with angiogenesis and growth factors, such as insulin-like growth factor binding protein 1 (IGFBP-1). 14 – 16 thus, as gestation nears term, the fetal liver is progressing from a hemopoietic to a metabolic function in preparation for postnatal life.Difference between hypoxia and anoxia

Fetal hypoxia can lead to local tissue formation of reactive oxygen species (ROS) such as superoxide anions, hydrogen peroxide, lipid peroxides, and hydroxyl radicals. 2, 17 excess generation, which may lead to oxidative stress, can result from a variety of sources that include nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, mitochondrial dysfunction, and reduced antioxidant capacity. 18 oxidative stress has been reported to contribute to cellular injury in a variety of organs such as brain, 19 heart, 20 and liver. 21 an excess of ROS generation that surpasses its antioxidant capacity has damaging effects that disrupt plasma membrane integrity via lipid peroxidation 20, 22 and nuclear oxidative damage via DNA fragmentation. 20, 21 chronic hypoxia impairs carbohydrate metabolism 11 and insulin signaling 23 in the fetal sheep liver, having a programming effect on insulin resistance in the offspring. 24 we propose that fetal hypoxia is likely to have a significant impact on normal liver function in utero via oxidative stress, contributing to altered fetal growth and metabolism.Difference between hypoxia and anoxia

The objectives of the study were to (1) quantitate the effect of chronic hypoxia on the fetal liver in generation of oxidative stress as an important stimulus for inducing fetal liver damage and (2) to test the protective effect of maternal administration of an antioxidant, N-acetylcysteine (NAC), against the fetal liver damage in the hypoxic fetus. This was assessed by measuring the tissue levels of malondialdehyde (MDA) as an index of lipid peroxidation and DNA fragmentation as an index of nuclear damage and apoptotic cell death.

Animal treatment and sample preparation

Pregnant guinea pigs (dunkin-hartley, term = 65 days) were placed in either a hypoxic (HPX) chamber (10.5% O 2 for 14 days prior to fetal excision, N = 6) or room air (normoxic or NMX, N = 5-7) as previously described. 25, 26 the selected gestational period is a rapid growth phase in fetal guinea pig development. 27 the hypoxic level (10.5% O 2 ) was the minimal oxygen level that could be imposed without causing pregnancy termination.Difference between hypoxia and anoxia these conditions generate fetal hypoxia as evidenced by increased RBC content and reticulocyte number in fetal guinea pig plasma, 28 increased hypoxia inducible factor 1α (HIF-1α) protein levels in hypoxic fetal cardiac ventricles, 29 and increased staining of hypoxyprobe 1 in hypoxic fetal livers. 30 in addition, a separate group of HPX and NMX animals were treated with NAC (500-600 mg/kg per d) by placing it in their drinking water for 10 days during the hypoxic period or during the age-matched gestational period in NMX animals, respectively. The dose was based on studies shown to inhibit an inflammatory cytokine response to LPS in rats 31 and effects of ROS in guinea pigs. 32 also, we have reported that NAC inhibits MDA levels in fetal guinea pig heart ventricles. 33 both food (g/d; tekblad guinea pig diet, harlan laboratories, frederick, MD) and water volume (ml/d) intake rates were measured throughout the duration of treatment.Difference between hypoxia and anoxia

At near term, 63 days of gestation (term = 65 days), pregnant sows were anesthetized (ketamine, 1 mg/kg; xylazine, 80 mg/kg) and fetuses were delivered by hysterotomy. Fetal body, liver, placenta, brain, and heart weights were measured. Right liver lobes were excised and either fixed for staining or snap frozen in liquid nitrogen and stored at −80°C. The study protocol was approved by university of maryland animal care and use committee and conforms to the guide for the care and use of laboratory animals published by the US NIH publication no. 85-23, 1996.

Fetal guinea pig body and organ weights

The effect of chronic hypoxia and NAC treatment on fetal body weight and liver weight is shown in figure 1.Difference between hypoxia and anoxia the average fetal body weight of NMX fetal guinea pigs was 88.8 ± 4.5 g. Hypoxia significantly decreased ( P .01) fetal body weight (59.5 ± 2.4 g) by 33% compared to NMX controls. Maternal administration of NAC had no effect on fetal body weight under conditions of either normoxia (88.8 ± 4.5 g vs 76.6 ± 7.8 g; NMX vs NMX-NAC) or hypoxia (59.5 ± 2.4 g vs 59.8 ± 2.4 g; HPX vs HPX-NAC). Mean liver weight of NMX fetuses was 4.59 ± 0.41 g. Hypoxia reduced liver weight (2.88 ± 0.18 g) compared to its NMX controls by 37.2% ( P .05). Mean fetal liver weights of NMX-NAC and HPX-NAC were 3.96 ± 0.42 g and 3.21 ± 0.18 g, respectively. When normalized to its fetal body weight, relative liver weights were similar among all groups (0.048 ± 0.002, 0.049 ± 0.003, 0.052 ± 0.001, and 0.054 ± 0.002 for NMX, HPX, NMX-NAC, and HPX-NAC group, respectively).Difference between hypoxia and anoxia chronic hypoxia increased ( P .05) the relative weights of placenta, brain, and heart ( figure 2), demonstrating a brain and heart sparing effect of hypoxia. Chronic hypoxia did not reduce the absolute weight of the placenta (4.69 ±0.24 vs 4.59 ± 0.33 g) although significantly ( P .05) reduced brain and heart weights compared to NMX controls (2.62 ± 0.06 vs 2.35 ± 0.06 g; 0.46 ± 0.03 vs 0.38 ± 0.01 g, HPX vs NMX, respectively).

Effect of chronic intrauterine hypoxia on fetal liver growth

Chronic hypoxia induces a decrease in fetal body weight associated with an increase in the relative placenta weight as previously described. 28 there was no change in the relative fetal liver weight despite hypoxia-induced changes in lipid peroxidation and DNA fragmentation.Difference between hypoxia and anoxia further, NAC did not reverse fetal growth restriction despite the ability of NAC to reverse lipid peroxidation and DNA damage in the fetal liver.

Chronic hypoxia reduces liver weight in proportion to fetal body weight. The changes in liver weight of hypoxic fetuses is likely the result of either poor oxygenation due to perfusion heterogeneity or oxygen-sensitive mechanisms 13, 35 that are unaffected by NAC directly since NAC had no effect on absolute liver weights or liver/body weight ratios. Perfusion of the liver has a direct relationship to its growth and function. 36 in human fetuses, fetal liver perfusion is met by 80% of blood flow from umbilical vein and 20% of splanchnic origin. 37 of the umbilical blood flow, 20% to 50% bypasses the fetal liver and is directed toward the inferior vena cava by the ductus venosus. 6, 38 fetal hypoxia diverts blood away from the fetal liver as an adaptive response for redistributing blood flow to the heart and brain. 39 – 41 this makes the fetal liver vulnerable to hypoxic stress as a result of redistribution of oxygen away from hepatic tissue.Difference between hypoxia and anoxia in contrast to human fetuses, the fetal guinea pig lacks a functional ductus venosus. 42, 43 in growth-restricted guinea pig livers, total hepatic blood flow is reduced and flow is distributed away from the right lobe preferentially. 44 thus, the hypoxic guinea pig liver (ie, right lobe) experiences a similar flow limitation as the hypoxic human liver with a ductus venosus. In the long term, the proportional decrease in liver growth is likely to have important implications in normal growth and metabolism of the fetus as well as contribute to neonatal morbidity via programming mechanisms.

In contrast to fetal liver, the relative placenta weight was increased by hypoxia and reversed by NAC. Despite the lack of changes in relative brain and heart weights by NAC, the effect of NAC on placenta differs from the fetal liver, brain, and heart.Difference between hypoxia and anoxia this may reflect the difference of NAC on factors modifying placental growth and/or the effect of oxidative stress in the hypoxic placenta.

Effect of hypoxia on cellular integrity of fetal liver

The fetal liver plays an important role in hemopoiesis as the primary site during fetal development. 7, 8 initially, the yolk sac blood islands are responsible for hemopoiesis during embryogenesis. During fetal development, progenitor cells seed the fetal liver, which then becomes the predominant site. 45 hepatocytes make up the majority of the parenchymal tissue, which are derived from terminal differentiation of progenitor cells. 7, 8 in response to hypoxia, stress erythropoiesis, a response that generates an increase in erythrocytes under conditions of reduced oxygen supply, 46 is induced in the fetal liver.Difference between hypoxia and anoxia we have previously measured an increase in plasma RBC content and reticulocyte count in hypoxic versus normoxic fetal guinea pigs under similar conditions. 28 in the current study, chronic hypoxia increases aggregation of erythroid progenitor cells in fetal guinea pig liver, suggesting a compensatory response to reduced oxygenation. The decrease in aggregate hemopoietic cell clusters following NAC administration suggests a response to oxidative stress and not to reduced oxygenation since hypoxic conditions still remain with NAC as evidenced by sustained heart and brain sparing effects. Since NAC did not completely eliminate this effect, it is possible that a combination of both oxidant molecules and other hypoxia-related mechanisms (ie, HIF-1α expression) 47 may contribute.Difference between hypoxia and anoxia

Oxidative stress induces cellular damage to molecules such as lipids, proteins, and DNA. As a result, phospholipids of plasma membranes are vulnerable to peroxidation by superoxide anions. 48 superoxide anions can induce marked fragmentation of nuclear DNA. 48, 49 defense mechanisms against oxidative stress is dependent on the relative activities of cu/zn superoxide dismutase (SOD) and mn-SOD. Fetal liver expresses lower levels of antioxidant enzymes compared to adult liver, 50 – 53 suggesting a reduced antioxidant capacity. 47 the fetal liver consists mainly of hemopoietic (or erythroid progenitor) cells during the early fetal period and increases in cell population to hepatocytes, sinusoidal cells, and bile duct cells later in gestation. 54 hemopoietic cells but not hepatocytes have been shown to exhibit marked DNA fragmentation as gestation progresses to term. 55 as term approaches, there is a progressive change in fetal liver cell composition due to rapid hepatocyte proliferation and regression of hemopoietic cells via apoptosis. 55 in late gestation, it is possible that oxidative stress may affect liver function in a cell-specific manner with a greater proportion of lipid peroxidation and DNA fragmentation occurring in hepatocytes.Difference between hypoxia and anoxia thus, intrauterine oxidative stress may alter the progression pattern of vulnerable cell populations and contribute to fetal liver dysfunction. The discrepancy between reversal of liver cell damage by NAC but not liver weight may indicate that only a limited fraction of the total liver is affected by hypoxia.

Our data do not indicate that NAC has direct adverse effects on the hepatic hemopoietic cells as demonstrated by the lack of histopathology and DNA damage in NMX livers. Further, the overall hemopoietic function may not be damaged by fetal hypoxia since red blood cells (rbcs) and their progenitor, reticulocytes, are increased in fetal plasma in response to hypoxic stress. 28 the role of the bone marrow as an additional hemopoietic system under these conditions is unclear.Difference between hypoxia and anoxia it is possible that chronic hypoxic stress could also trigger erythropoiesis of the bone marrow as a compensatory response.

Effect of NAC on hypoxia responses

Reactive oxygen species such as superoxide anions, hydrogen peroxide, and lipid peroxides can play both a physiological and pathological role in the fetal liver. When generated in excess, these oxidant molecules can contribute to fetal toxicity by their interactions with cell organelles within specific fetal organs. N-acetylcysteine is an antioxidant that crosses the placenta and has previously been shown to inhibit oxidative stress during pregnancy. 56 – 60 in the current study, the hypoxic fetal guinea pig liver exhibits histopathological damage, increased lipid peroxidation, and DNA fragmentation.Difference between hypoxia and anoxia

Malondialdehyde is a by-product of lipid peroxidation generated in the presence of oxidant molecules such as superoxide anions and used to assess hypoxic stress and the effectiveness of antioxidants such as NAC. Oxidative stress generated by increased superoxide anions can directly cause DNA breaks, which may contribute to the increased fragmentation levels. Additionally, there may be indirect effects of oxidative stress that alter the ability of DNA to repair DNA breaks. The protective effect of maternal NAC treatment on the hypoxic fetal liver is supportive of oxidant damage. In cultured fetal rat liver cells, exogenously added NAC, reduced glutathione (GSH), or l-cysteine inhibited the DNA fragmentation that occurs in the perinatal period in hemopoietic cells but not hepatocytes. 55 yet, addition of superoxide dismutase or catalase enzymes that decrease superoxide anion and hydrogen peroxide accumulation, respectively, did not reverse the DNA damage in these same cells.Difference between hypoxia and anoxia thus, antioxidant thiols have a protective role against hemopoietic cell injury, which may identify a cell-specific, and perhaps a ROS-specific, response to oxidative stress.

It is possible that NAC affects hypoxia signaling rather than reversing oxidative stress. This is supported by evidence in cultured cancer cells showing that NAC inhibits HIF-1α expression. 61, 62 however, there is no direct evidence that NAC affects either hypoxia signaling or oxygen sensing (ie, chemoreceptor cells) in vivo in the fetus. 63 lastly, while the direct effect of NAC on uteroplacental blood flow was not measured in this study, it is likely to be insignificant due to the persistence of fetal hypoxia in the presence of NAC administration.Difference between hypoxia and anoxia

In conclusion, this study identifies the fetal liver as sensitive and vulnerable to reduced oxygenation and that oxidative stress plays a causal role in fetuses exposed to chronic intrauterine hypoxia. This is mediated by impairment of cell membrane integrity by lipid peroxidation and DNA fragmentation. The impact of oxidative stress on fetal liver damage may have several consequences including contributing to fetal growth restriction and altering both the hemopoietic response to hypoxia 64 and the cellular integrity of the fetal liver. The protective effect of NAC confirms the damaging effects of oxidative stress in the hypoxic fetal liver and indicates that antioxidants may be a potential therapeutic approach for the prevention of subsequent fetal and neonatal morbidity associated with chronic intrauterine hypoxia.Difference between hypoxia and anoxia