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1 Veterans Affairs Medical Center, Pittsburgh 15240; Departments of 2 Medicine and 3 Surgery, and 4 Center for Genomic Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; and 5 Mirus Corporation, Madison, Wisconsin 53719
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
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Sex differences in susceptibility to alcohol-induced liver injury have been observed in both humans and experimental animal models. Using a standard model of alcohol-induced fatty liver injury and microarray analysis, we have identified differential expression of hepatic genes in both sexes. The genes that exhibit differential expression are of three types: those that are changed only in male rats fed alcohol, those that change in only female rats fed alcohol, and those that change in both sexes, although not always in the same manner. Certain of the differentially expressed genes have previously been identified as participants in the induction of alcohol-induced liver injury. However, this analysis has identified a number of genes that heretofore have not been implicated in alcoholic liver injury; such genes may provide new areas of investigation into the pathogenesis of this disease.
alcohol; microarray analysis
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
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ALCOHOL-INDUCED LIVER INJURY (ALI) and disease (ALD) are major health problems affecting a broad patient population of different gender, race, and social backgrounds. The present model of pathogenesis of ALI exploits the role of different cytokines acting by induction of various transcription factors that in turn modulate expression of different genes through action on appropriate response elements and binding sites on those genes. Alcohol consumption triggers intrahepatic events such as alcohol metabolism in hepatocytes, which can lead to oxidative and metabolic stress. Extrahepatic events can also be involved, such as release of gut-derived endotoxin into the circulation, which acts on Kupffer cells in the liver. Both of these types of events can initiate and perpetuate an interplay between different functional cell groups in the liver, i.e., hepatocytes, Kupffer cells, hepatic stellate or Ito cells, mononuclear cells, and neutrophils, resulting in fatty liver, necrosis, inflammation, and fibrosis seen in ALI (reviewed in 15, 16, 30, 33, 52).
In humans, significant gender differences in susceptibility to ALI are observed (31, 38). Women appear to be at greater risk for more severe ALI, in that they develop cirrhosis more quickly and with a lesser alcohol intake than do men. Some evidence from certain rat models of alcohol ingestion, i.e., the Tsukamoto-French intragastric model, shows similar female susceptibility in degree of ALI (25), but this is not a universal finding, and only very rarely have direct side-by-side comparisons between male and female rats been performed.
One of the molecular mechanisms responsible for such a sex difference in susceptibility of liver to ethanol may be caused by differential expression of hepatic genes, especially those regulated by sex hormones. These genes encode for proteins responsible for the cellular responses to a toxic stress such as the chronic consumption of ethanol. Hence, a broad screen of hepatic gene expression in animals chronically fed ethanol is necessary to achieve a greater insight into the mechanisms of cellular adaptation or the failure thereof that leads to ethanol-induced liver injury.
Development of cDNA expression arrays (microarrays) provides a new powerful tool to perform a gene-expression screen of hundreds or thousands of genes in a single hybridization (45). The study described here was designed to profile the expression of hepatic genes in both sexes in response to a chronic ethanol diet. The model of chronic ethanol feeding used in this study (Lieber-DeCarli) induces a spectrum of biochemical changes and fatty liver with minimal or no inflammation or fibrosis after 30 or more days of exposure. Use of this model is of particular interest to obtain an insight into early changes in gene expression and sex differences as a response to ethanol under such experimental conditions, because fatty liver can proceed to more severe ethanol-induced liver injury. For our gene expression studies, we used a commercially available microarray designed to screen 465 genes that are known to be related to toxic stress.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
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Alcohol Feeding Protocol
Liver samples were obtained from animals fed using the Lieber-DeCarli model (42). Male and intact female Wistar rats (Charles River, Wilmington, MA), beginning at 35-36 days of age, were divided into an alcohol-fed group with 36% of total calories as alcohol (AF) and a control group pair-fed with an isocaloric, nonalcoholic diet (IC); both groups were fed for 30-42 days, a period sufficient to develop alcoholic fatty liver. The liquid diets were obtained from BioServe (Frenchtown, NJ) and reconstituted according to directions. Intake was ad libitum for the alcohol-fed rats, and their pair-fed controls received the exact amount that the alcohol-fed animal consumed the previous day, a protocol that eliminates differences in caloric intake. Animals were killed after 30 and 42 days by ether inhalation and exsanguination via the abdominal aorta. Livers were removed, weighed, and immediately frozen at
80°C
for later analysis. Serum was prepared from blood by standard
centrifugation methods. This animal model has also been used in our
previous work (14, 50, and references therein).
Histopathological Analysis
Livers were cut into 4-µm sections and stained with hematoxylin and eosin, after which liver injury was scored blindly. Although all hepatic tissue for any given liver showed similar morphology, different hepatic units in the same section could show different scores of pathology. To be as objective in scoring as possible, both sections in slides were examined, and an average bulk score for each slide was assigned. The scoring system for grading the hepatic pathology is as follows.Hepatic steatosis. Hepatic steatosis was defined in hematoxylin- and eosin-stained sections as the presence in hepatocyte cytoplasm of colorless vesicles/vacuoles with very sharp borders. Because there is a lack of uniform agreement in the literature for morphological grading of hepatic steatosis (23), we have adopted with minimal modifications several scoring systems, from both clinical (17, 23, 32, 44) and experimental (7, 24) studies, as follows. Score 0: normal liver morphology, no fat drops. Vacuolization of hepatocyte cytoplasm, if present, does not show a sharp border of vacuoles. Score 1: fat vesicles, mostly macrovesicles, present in scattered hepatocytes, with no identified pattern, probably tending to zones 3-2 of hepatic unit. Score 2: fat vesicles, mostly macrovesicles, present in scattered hepatocytes predominantly in zone 3 of hepatic unit, affecting 10-30% hepatocytes of zone 3. Score 3: fat vesicles, macro- and microvesicles, present in 50% or more hepatocytes in zone 3 of hepatic unit, forming areas of affected parenchyma. Score 4: fat vesicles, macro- and microvesicles, present in most of hepatocytes in zones 3 and 2 of hepatic unit, forming areas of affected parenchyma and sometimes forming bridging connection between hepatic lobules. Score 5: fat vesicles, macro- and microvesicles, present in hepatocytes of all zones of hepatic unit, forming areas of affected parenchyma that always forms bridging connection between hepatic lobules.
Hepatic necroinflammatory damage. The scoring system for grading hepatic necroinflammatory damage was modified from several reports on clinical (2, 22) and experimental (10, 35, 43) studies. The main difference in our model was an insignificant portal inflammation and damage to periportal hepatocytes. The extent of hepatic inflammation in our study was not significant; therefore the highest score reported still corresponds to mild/moderate inflammation. Score 0: normal liver morphology or presence of single inflammatory cells or rare small cell collection without consistent parenchymal damage. Score 1: presence of scattered inflammatory cells associated with hepatocyte damage/dropout. Score 2: presence of scattered small inflammatory cell collections associated with hepatocyte damage/dropout; small portion of hepatic units affected. Score 3: presence of small inflammatory cell collection in majority of hepatic units associated with hepatocytes damage/dropout. Score 4: presence of more than one inflammatory cell collection in majority of hepatic units associated with hepatocyte damage/dropout.
Hepatic fibrosis. The extent of hepatic fibrosis in our study was minimal and was scored as following. Score 0: normal liver morphology with presence of single collagen fibers around portal areas and big hepatic veins but not invading hepatic parenchyma. Score 1: single collagen fibers are present around majority of small-medium hepatic veins but do not invade into hepatic parenchyma. Score 2: single collagen fibers are present around majority of small-medium hepatic veins sometimes invading hepatic parenchyma and forming bridges between approximated central veins.
Serum Testosterone and Estradiol
Methods for the radioimmunoassay for serum testosterone have been previously published (11-13). Serum estradiol determinations were performed by the Radioimmunoassay Core, Center for Research in Reproductive Physiology, University of Pittsburgh School of Medicine.RNA Isolation
Total hepatic RNA was extracted from frozen liver by using a standard guanidium thiocyanate phenol-chloroform extraction method (8) and was treated with the RNase-free DNase I provided in the microarray kit (Clontech, Palo Alto, CA) to reduce DNA contamination. Total RNA concentration and purity were determined spectrophotometrically, and the integrity of RNA samples was ascertained by appearance of distinct 28S and 18S bands of ribosomal RNAs on 1.2% agarose gel electrophoresis.Microarray Analysis
Microarray analysis was performed according to manufacturer's instructions (www.clontech.com) by using the Clontech Atlas Rat Toxicology II array. The 465 genes on the array represent various cellular functions that may be changed by toxic substances such as ethanol and include those for transcription factors, enzymes of common metabolic pathways, xenobiotic metabolism, receptors, cytokines, signaling molecules, immunomodulators, acute-phase proteins, antioxidant enzymes, and modifiers of protein synthesis and secretion, among others. The gene sequences were applied to a nylon membrane, with two separate spots for each gene. Each array also includes a set of housekeeping genes, together with negative control and genomic DNA. A complete list of the genes on this array is provided by the manufacturer (www.clontech.com). Each gene on the list is accompanied by the National Center for Biotechnology Information (NCBI) GenBank accession number of a published sequence of the gene and the sequence of related protein.A total amount of 4 µg of purified RNA was used for the hybridization of each single microarray membrane. To reduce variability of changes, probes were made by pooling RNA samples from two to four animals from control or ethanol-fed group of animals and repeating the same microarray analysis two to three times. In brief, purified RNA was converted to 32P-labeled cDNA probe by using the Moloney murine leukemia virus reverse transcriptase, master mix, and specific primer mix provided in the microarray kit (Atlas Rat Toxicology II). The cDNA probe was then purified by using G-50 Sephadex columns (Eppendorf 5-Prime, Boulder, CO), and intensity of radioactive labeling was measured by using a beta scintillation spectrometer. Microarray membranes were prehybridized with the kit's ExpressHyb solution containing heat-denatured salmon testes DNA (100 µg/ml of buffer) for >1 h, followed by a hybridization overnight at 68°C with the cDNA probes. After hybridization, membranes were washed with 2 different washing solutions [2X sodium chloride-sodium citrate (SSC), 1% SDS, 3 × 30 min, and 0.1X SSC, 0.5% SDS, 1 × 30 min] at 68°C, then soaked in 2X SSC solution for 5 min at room temperature, wrapped in plastic wrap, and exposed to a phosphoimaging screen. The image obtained by using the phosphoimager was then converted to a PDF file, and hybridization intensity was analyzed by using Clontech AtlasImage 1.5 software.
Real-time PCR
To verify and quantify the changes in expression of some of the genes obtained by microarray method, we used real-time PCR (TaqMan) as a complementary method. We chose three genes from each membrane [tyrosine aminotransferase (TAT), lipopolysaccharide-binding protein (LPS-BP), and calreticulin precursor (CALR)] for verification. Gene-specific primers were designed by using the Primer Express 1.0 software (PE Applied Biosystems, Foster City, CA) according to the NCBI accession numbers for published sequences from GenBank, provided by the genes list in the Clontech manual. Primer sequences for TAT were 5'-TGG AGT TCA CAG AGC GGT TG-3' (sense) and 5'-GGT ACT CGA AGC ACG TTG CTG-3' (antisense). Primer sequences for LPS-BP were 5'-TCA CTC ACC CAC GGA GCA T-3' (sense) and 5'-CCC CAG CAA TGT AGG AAG CA-3' (antisense). Primer sequences for CALR were 5'-CCA GAC AAC ACC TAC GAG GTG A-3' (sense) and 5'-GTC CCA ATC ATC CTC CAA GGA-3' (antisense). All primer pairs were purchased from GIBCO BRL Custom Primers, Life Technologies (Rockville, MD). Total liver RNA was used from the same control and ethanol-fed animals used in the microarray hybridization experiments. The primers used for the quantification of rat 18S rRNA (GenBank accession no. M11188) as a standard internal control were purchased from Integrated DNA Technologies (Coralville, IA); the rat 18S1 sequence was 5'-GAG GCC CTG TAA TTG GAA TGA GTC-3' and rat 18S2 sequence was 5'-TCC CAA GAT CCA ACT ACG AGC TTT-3'. SYBR green PCR reagents (PE Applied Biosystems) were used for real-time PCR, and PCR reaction was run by using the ABI PRISM 7700 sequence-detection system (PE Applied Biosystems). A more detailed description of the TaqMan protocol and conditions has been published (29, 41). Results were analyzed by using ABI PRISM 7700 Sequence Detection version 1.6.3 to obtain CT values, where CT values are threshold cycles at which a statistical significant increase in detection of SYBR green emission intensity occurs. Then, CT values are normalized to 18S endogenous control to account for variability in RNA concentrations between samples to obtain
CT values. To obtain
CT values, the CT value for the
control rat is subtracted from the CT value of the
alcohol-fed rat. Finally, the relative quantitation (r.q.) value is calculated as
Statistics
Microarray hybridizations were performed at least two times on separate arrays with samples derived from animals fed the same protocol. For normalization of individual hybrid intensities, we used the polyubiquitin housekeeping gene because it has shown to have the most consistent expression pattern compared with other housekeeping genes on the array. Hybridization intensity of repeated array experiments were expressed as means ± SE. Student's t-test was used to analyze the differences between control and ethanol-fed animals and between male AF and female AF animals, and a P value of <0.05 was considered to be significant.| |
RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
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As shown in Table 1, there was no
difference in the body weights of either male or female AF rats
compared with their same-sex controls. However, the livers of both male
and female AF rats weighed significantly more than those of their IC
controls and consequently demonstrated significantly increased liver
weight-to-body weight ratios (increased 30% in males and 29% in the
females, both P < 0.05), indicating the hyperplasia
typical of alcohol-fed animals.
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The results of the histopathological examination, shown in Table
2, indicate that both male and female AF
rats have fatty liver. However, in this study, male AF rats also
demonstrated inflammation and fibrosis and overall liver injury greater
than the female AF rats (inflammation and overall injury,
P < 0.05).
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Table 3 shows changes in serum sex
hormones in both the male and female AF groups compared with their
controls. The male AF rats have a significant increase in serum
estradiol levels (increased 63%, P < 0.05) and
decreased serum testosterone levels (decreased 80%, P < 0.05). Additionally, the male AF rats demonstrated testicular
atrophy, with testes weight/body weight decreased 15% compared with
their controls (AF mean ± SD, 0.0096 ± 0.0026 vs. IC
0.0114 ± 0.0025, P < 0.05). The chronic alcohol
feeding regimen resulted in a significant decrease in serum estradiol
levels in female AF rats compared with their IC rats (decrease 30%,
P < 0.05). The altered levels of sex hormones in the
alcohol fed rats of both sexes may reflect a delay in puberty in these
animals, because the rats were started on the alcohol diets at ~35
days, before puberty.
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Figure 1 displays representative images
of microarray membranes hybridized to radiolabeled cDNA probes derived
from RNA of control and ethanol-fed male rats. Using such a gene
expression screen tool that is geared toward detecting toxicological
changes, we found a number of genes whose expression is altered in rats of both sexes fed ethanol chronically. Protein products encoded by
these genes belong to a broad group of various functional proteins that
may be related to cellular functions altered by ethanol as a toxin,
such as xenobiotic metabolism and metabolism of carbohydrates, proteins
and lipids, as well as function of transcription factors, antioxidant
enzymes, and modulators of protein trafficking and secretion, among
others. Although some of the changed genes and their related protein
products have been previously identified as involved in the
pathogenesis of ethanol-induced liver injury, most of the genes, to
date, have not been clearly related to ethanol effects.
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Because the specific intent of this study was to investigate sex
differences in response to chronic ethanol exposure, we addressed changes in gene expression in young male or female rats, with particular emphasis on genes that may be regulated by sex hormones. Not
only do the sexes differ with respect to hormonal levels, but chronic
alcohol exposure is known to alter serum sex hormone levels, as shown
in Table 3 and Refs. 13 and 14. We have sorted gene
expression data into three distinct groups: first, genes changed only
in male rats fed ethanol (Table 4, Fig.
2); second, genes changed only in
female rats fed ethanol (Table 5, Fig.
3); and third, genes changed in rats
of both sexes (Fig. 4), although not
always in the same direction.
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Gene Expression Changes in Male Rats
The first group of genes represented in Table 4 is the one that changed in male rats fed ethanol compared with its pair-fed isocaloric controls. There were 38 genes changed in male rats fed ethanol, with a range of change from 1.77-fold to more than 25-fold. The profile of the whole group of genes outlines the complexity of changes in liver induced by ethanol. The genes in this group encode for proteins that belong to various functional groups such as transcription factors (hepatocyte nuclear factor-4
), xenobiotic metabolism (glutathione
S-transferase Yc1 subunit, catechol-O-methyltransferase, cytochrome
P-450 2B1, alcohol-sulfotransferase, cytochrome
P-450 IIC9, heat shock 90-kDa protein-
,
UDP-glucuronosyltransferase 2B); metabolism of lipids
(long-chain-specific acyl-CoA dehydrogenase precursor,
apolipoprotein-A1 precursor), proteins (cytoplasmic aspartate
aminotransferase, tyrosine aminotransferase), and carbohydrates [phospho(enol)pyruvate carboxykinase, IGF-1B precursor]; protein synthesis and secretion modifiers [protein disulfide isomerase, 40S
ribosomal protein S30, ubiquitin-like protein (NEDD8), tissue inhibitor
of metalloproteinase-1 precursor gene (TIMP-1), NADPH-cytochrome P-450 reductase], acute-phase proteins or those involved in
immunological response (complement component 4-binding protein-,
mitochondrial stress 70 protein precursor, -1 acid glycoprotein,
T-cell cyclophilin). Some of those genes and related proteins have been
already known as associated or changed by ethanol effects (IGF-1B and
IGF-1 binding protein, cytoplasmic aspartate aminotransferase, retinoid X receptor ) or involved in pathogenesis of ALI (TIMP-1, LPS-BP). All other genes encode for functional proteins involved in processes that may be related to early biochemical and metabolic changes induced
by ethanol that further may proceed to later morphological damage.
Furthermore, some of the genes changed only in male rats fed ethanol
are regulated by androgens (C4-BP, carbonic anhydrase 3, CYP2B1,
Apo-A1, MB-COMT, NADPH-CYP reductase, IGF-1B, TIMP-1), further
suggesting that a modifying or regulatory role of sex hormones
(androgen or estrogen) on hepatic functions may change in response to
chronic ethanol exposure, and this change may create sex-related
difference in susceptibility to ethanol. For example, the gene that
encodes for carbonic-anhydrase III is downregulated in AF male rats and
is testosterone dependent (3). The reduction of expression
may represent a loss of a potential "hepatoprotective" function of
androgen, because the product of this gene has been suggested recently
as a potential protective factor against oxidative stress. In
particular, this protein protects against oxidative stress-induced
apoptosis (39), and an increase is associated with
decreased aldehydic protein adducts and minimal histopathology in male
micropigs fed ethanol (40).
Gene expression of another androgen-regulated gene, apolipoprotein-A1 precursor (28, 51), was upregulated in our study of male rats, suggesting that change of the lipoprotein profile in chronic male alcoholics may be attributed to an ethanol-induced alteration of hormonal control of lipoprotein metabolism and/or synthesis. Another example is the finding of an overexpression (10-fold) of CYP2B1 gene induced by ethanol. This gene is normally expressed in very low levels in both sexes but is also highly induced by phenobarbital only in male rats and under a strong regulatory role of androgens (4). Overexpression of this particular xenobiotic pathway may be a part of an overall induction of xenobiotic metabolism induced by ethanol that serves as a detoxification pathway but one that could also create potentially harmful effects or metabolites.
An intriguing finding may be an upregulation of NADPH-cytochrome P-450 reductase gene in ethanol-fed male rats, also influenced by androgens (5). It has been shown recently that this enzyme is implicated in the short cellular half-life of CYP2E1 and thus may limit the activity of this enzyme as a known pathway of reactive oxygen species generation in ALI (58).
Two genes related to IGF-1, IGF-1B precursor and IGF-binding protein-1 precursor gene, were upregulated in ethanol-fed male rats in our study. It has been shown that IGF-1 mRNA and protein levels are reduced in ethanol-fed animals, whereas mRNA levels for IGF-binding proteins are increased (27). Testosterone may increase the IGF-1 circulatory levels, contrary to effects of ethanol on IGF-1 (59), suggesting an interesting link to a mechanism of greater susceptibility of male sex to alcohol-induced myopathy, because skeletal muscle is a target tissue to positive effects of IGF and serum testosterone levels are decreased in ethanol fed male rats and humans. However, without an analysis of the protein products, it is impossible to determine whether the induction of these two genes impacts on the bioavailability of IGF-1.
Although epidemiological studies have shown greater overall susceptibility of female sex to ethanol-induced liver injury (38), there may be specific sex differences for certain of the effects of ethanol, so that each sex might have susceptibility to a specific type of injury. For example, the finding of an upregulation of TIMP-1 gene in AF male rats may suggest their potentially greater susceptibility to liver fibrosis and progression to cirrhosis, because this TIMP-1 inhibits cellular breakdown of collagen. This theory is supported by a recent study showing an antifibrotic effect of estrogen-induced downregulation of TIMP-1 (57).
Gene Expression Changes in Female Rats
Genes changed only in female rats fed alcohol, compared with their isocalorically pair-fed controls, are shown in Table 5. Some of these genes are known to be regulated or affected by estrogen levels: apolipoprotein E precursor, cytochrome P-450 2E1, copper-zinc-containing superoxide dismutase-1, liver catalase, and C-reactive protein. A downregulation of their expression in this study may suggest that the physiological regulatory effects of estrogen on hepatic functions may be decreased after chronic ethanol consumption, and indeed estradiol levels are lower in the AF rats in this study. Some of those regulatory functions may have a protective effect, and their decrease may create greater susceptibility of female sex to ethanol-induced effects. For example, the alcohol-induced downregulation of apolipoprotein E precursor gene, which is regulated by estrogen via estrogen receptors (48), may suggest a possible reduction of antiatherogenic apolipoprotein E and a change to a more atherogenic (i.e., male) lipid profile. This is supported by recent findings that overexpression of this gene has a protective effect in development of atherosclerosis in mice (49). Another example of the changes in females is the downregulation of liver catalase gene, the protein product of which metabolizes reactive peroxides. Such a downregulation may account for a reduced hepatic antioxidant defense in female rats exposed to ethanol for a longer time period. Our findings correlate with the study in which reduced catalase activity in liver was found after decreased gonadal production of estrogen (47). Expression of the copper-zinc-containing superoxide dismutase-1 gene was upregulated in the AF female rats; this finding may reflect the decrease of serum estrogen levels or possible alteration in estrogen receptor function, because an increased activity of this enzyme was also shown after oophorectomy (21). However, the potential pathological significance of this change is less clear.We have also found a decreased expression of the CYP2E1 gene in AF female rats. This result may suggest a diminished estrogen regulatory role or decreased activity of estrogen receptors in the liver, because it has been shown that CYP2E1 enzyme activity correlates directly with serum estrogen levels, i.e., increased by estradiol administration (1, 36). It is not clear whether this change of CYP2E1 gene expression may have an effect on CYP2E1 enzyme activity and whether a subsequent protective or negative effect may result.
Expression of the gene that encodes for the synthesis of C-reactive protein is also downregulated in AF female rats. The function of this acute-phase protein as an immunomodulator and evidence of its regulation by estrogen may suggest a greater susceptibility of female rats to ethanol-induced inflammatory liver damage (11, 37).
Gene With Changed Expression in Both Sexes of Ethanol-Fed Rats
The third group of genes is the one for which expression was changed in both sexes, male and female rats (boldface gene names in Tables 5 and 6 and Fig. 4), although the pattern of expression change was sex-different in two-thirds of the genes. Among the three genes that showed the same pattern of change (upregulation in both sexes in AF rats), two are known to be regulated by androgen [IGF binding protein-1 precursor gene and phosphoenolpyruvate carboxykinase] (55), whereas the tyrosine aminotransferase gene has no sex differences in regulation of its protein product activity (42). Because phosphoenolpyruvate carboxykinase catalyses the rate-limiting step of hepatic gluconeogenesis, this may represent a molecular mechanism for a higher susceptibility of male rats to alcohol-induced alteration in glucose metabolism and the hypoglycemia associated with ethanol consumption. Alternately, this finding may suggest the ability of the alcohol-fed males to compensate metabolically to increase serum glucose concentrations.
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Six genes were changed in AF rats of both sexes but with different patterns of change: increased expression in male rats and decreased expression in female rats (Tables 4 and 5, boldface gene names). Functions of the protein products of these genes suggest that there may be sex differences in susceptibility and especially a potentially greater susceptibility of female rats to ethanol diet in regard to various liver functions altered by ethanol. The genes for two enzymes involved in xenobiotic metabolism, UDP-glucuronosyltransferase gene (18) and alcohol sulfotransferase gene (46, 54), are downregulated in AF female rats, suggesting a sex difference and a potentially greater susceptibility of female rats to effects of ethanol on various enzymatic pathways involved in the process of detoxification. Alcohol sulfotransferase gene is also more highly expressed in untreated female than in male rats, and its downregulation in female rats fed ethanol may reflect ethanol-induced changes in sexually dimorphic liver function and "switching" of the normal sexual phenotype of the liver and other organs, as observed previously in chronic (male) alcoholics.
The gene for precursor for endoplasmic reticulum stress protein 72 is also diversely regulated in AF rats depending on sex (upregulated in male and downregulated in female rats). This protein has a protein disulfide isomerase activity and is involved in regulation of protein secretion (53), suggesting that some protein trafficking functions may be more profoundly impaired in female rats. There is also a significant downregulation of the dual-specificity mitogen-activated protein kinase kinase 2 gene in female rats, which may result in diminished ability of AF females to initiate liver regeneration (6).
One very intriguing finding in our study is the diverse expression pattern of LPS-BP gene. LPS-BP is essential for rapid induction of an inflammatory response when a small amount of LPS is present. Recent studies have generated new insight into LPS kinetics and suggest a protective role of LPS-BP (21), contrary to previous beliefs (20, 56). The significant upregulation of LPS-BP in male rats and the opposite trend in female rats may suggest greater susceptibility of female sex to inflammatory effects of chronic ethanol consumption and may help explain the previous observation that alcohol-induced liver injury in females develops more quickly and after less amount of alcohol (19, 31, 38).
Verification of Microarray Results By Real Time RT-PCR
Figure 4 depicts a logarithmic amplification plot for real-time RT-PCR quantification of mRNA levels of hepatic tyrosine amino transferase in AF and IC male rats, using gene-specific primers, as well as the plot for the normalization standard 18S rRNA. As shown in Fig. 5 and Table 6, three genes were verified in several different AF and IC rat pairs of both sexes (TAT, LPS-BP, and CALR). As noted in Table 6, this technique verifies the direction and extent of the changes observed by microarray analysis, although the values for the fold increase or decrease are not necessarily equivalent.
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Possible Mechanisms of Sex-Specific Susceptibility to Ethanol-Induced Liver Damage and Other Effects of Ethanol
The pattern of change of genes regulated by sex hormones in AF animals shows that regulatory role of sex hormones may change as a result of ethanol exposure and that such change may be protective or harmful, depending on previously "protective" or "inhibitory" role of sex hormone in a specific physiological instance. The effects of the sex steroids on various liver functions are mediated through hepatic sex hormone receptors (cytosolic and nuclear). Ethanol may affect hormonal regulation of hepatic function by three mechanisms: 1) by decreasing the gonadal production of the hormone and thus the serum levels of the hormone and overall bioavailability; 2) by affecting the function of sex hormone receptors, through the effects on protein synthesis or activity, or 3) by changing the sex hormone metabolism in the liver. All three proposed mechanisms may affect regulatory role of the major sex hormone, and sexual dimorphism may change or "switch" to opposite-sex phenotype. That switch in some of hepatic dimorphic functions may proceed to further morphological damage induced by ethanol. One of such examples is the change in secondary sexual characteristics in chronic alcoholics that accompanies or may precede further development of ALD. Our previous studies identified changes in all of the above parameters. In particular, males exposed chronically to alcohol develop a more female pattern of serum sex hormones and of liver gene expression. A striking example of the latter is the alcohol-induced loss of androgen-dependent estrogen metabolizing enzymes in male rats (14, 50). The changes noted in some of the genes in male rats, especially those that are under androgen control, may lead to a hypothesis that the major sex hormone, i.e., androgen in male rats and estrogen in female rats, may have an overall protective role.Our findings of gene expression changes for certain functions controlled by sex hormones make an ultimate hypothesis of greater susceptibility of female sex seem more complex. Our results may also imply that different sexes are more susceptible to a certain alterations induced by ethanol, such are greater susceptibility of female sex to xenobiotic metabolism and oxidative/inflammatory damage and potentially greater susceptibility of male rats to liver fibrosis. This may change the concept of the overall greater susceptibility of one, i.e., female, sex to ethanol-induced liver injury.
It should be emphasized that these preliminary findings represent changes in young rats, comparable to young adolescents in humans. Because the rats were started on the alcohol regimen before puberty, the changes noted may reflect an alcohol-induced delay in puberty. The possible consequence of such a delay is seen by the hormone levels in Table 3. If the female rats had been exposed to alcohol after puberty had occurred and normal estrogen levels were established, perhaps we would have observed more liver injury, as is typical of the adult women studied by others. Further, the potential effect of estrogen as a cofactor in alcoholic liver injury cannot be ignored in the young male rats in this study, given that they have very high estrogen-to-testosterone ratios, and thus their greater liver injury may be related to their estrogen levels.
Overall Conclusion Regarding Findings of This Study and Unanswered Questions
This is a novel type of gene expression study and a novel approach to study the regulatory role of sex hormones on liver functions. Although microarray analysis represent a powerful gene expression screening method with regard to the extent of information about the potentially changed genes in AF animals, it cannot address some of very important questions. First, one cannot conclude from the gene expression change the actual impact on synthesis and function of encoded protein product, and further, the impact of the ratio of change of gene expression on protein synthesis remains to be verified by studies of the protein products of the gene. Thus further complementary studies such as real-time quantitative PCR and protein and activity quantitation are needed to address further posttranscriptional and posttranslational events for each gene chosen for a specific mechanistic study. Lastly, it is not always obvious whether a change in expression of a given gene is a cause of, or caused by, the alcohol-induced liver pathology. However, the specific value of a study such as this is that the analysis gives valuable initial information that may direct future studies of ethanol-induced liver injury. Furthermore, it draws more attention to genes that have not been previously implicated in the pathogenesis of ethanol-induced liver injury, thus opening many possibilities for study of new genes and proteins and their potential role in ALI.| |
ACKNOWLEDGEMENTS |
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The authors are grateful for the advice and expertise of Bryan S. Thompson and Dr. Tony Godfrey of the University of Pittsburgh Center for Genomic Sciences, especially for help in the TaqMan analysis. The authors thank Dr. Clifford Pohl, Director of the Radioimmunoassay Core, Center for Research in Reproductive Physiology, University of Pittsburgh School of Medicine, for coordinating the measurement of serum estradiol.
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FOOTNOTES |
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This work was supported by Veterans Affairs Merit Review awards (to D. C. Whitcomb and P. K. Eagon), National Institute of Diabetes and Digestive and Kidney Diseases award DK-50236 (to D. C. Whitcomb), and the Veterans Research Foundation of Pittsburgh. P. K. Eagon was the recipient of a Veterans Affairs Research Career Scientist Award during the period of this work.
Address for reprint requests and other correspondence: P. K. Eagon, Univ. of Pittsburgh School of Medicine, S-848 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (E-mail: pkeagon2{at}pitt.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 24, 2002;10.1152/japplphysiol.00568.2001
Received 4 June 2001; accepted in final form 19 May 2002.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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