U.S. patent application number 12/043666 was filed with the patent office on 2009-08-06 for primary rat hepatocyte toxicity modeling.
This patent application is currently assigned to OCIMUM BIOSOLUTIONS, INC.. Invention is credited to Arthur L. Castle, Michael R. Elashoff, Brandon Higgs, Kory R. Johnson, Donna L. Mendrick, Michael Orr, Mark W. Porter.
Application Number | 20090197258 12/043666 |
Document ID | / |
Family ID | 37662053 |
Filed Date | 2009-08-06 |
United States Patent
Application |
20090197258 |
Kind Code |
A1 |
Mendrick; Donna L. ; et
al. |
August 6, 2009 |
PRIMARY RAT HEPATOCYTE TOXICITY MODELING
Abstract
The present invention is based on the elucidation of the global
changes in gene expression and the identification of toxicity
markers in tissues or cells exposed to a known toxin. The genes may
be used as toxicity markers in drug screening and toxicity assays.
The invention includes a database of genes characterized by
toxin-induced differential expression that is designed for use with
microarrays and other solid-phase probes.
Inventors: |
Mendrick; Donna L.;
(Gaithersburg, MD) ; Porter; Mark W.;
(Gaithersburg, MD) ; Johnson; Kory R.;
(Gaithersburg, MD) ; Higgs; Brandon;
(Gaithersburg, MD) ; Castle; Arthur L.;
(Gaithersburg, MD) ; Orr; Michael; (Gaithersburg,
MD) ; Elashoff; Michael R.; (Gaithersburg,
MD) |
Correspondence
Address: |
COOLEY GODWARD KRONISH LLP;ATTN: Patent Group
Suite 1100, 777 - 6th Street, NW
WASHINGTON
DC
20001
US
|
Assignee: |
OCIMUM BIOSOLUTIONS, INC.
|
Family ID: |
37662053 |
Appl. No.: |
12/043666 |
Filed: |
March 6, 2008 |
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Application
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10357507 |
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12043666 |
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60353171 |
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60363534 |
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60371134 |
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60371150 |
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60371413 |
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60374139 |
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60394253 |
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60378652 |
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60373602 |
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60378653 |
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60378665 |
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60407688 |
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Current U.S.
Class: |
435/6.16 |
Current CPC
Class: |
C12Q 1/6876 20130101;
Y10S 707/99945 20130101; C12Q 2600/158 20130101; G01N 33/5014
20130101; Y10S 707/99948 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of determining whether a compound induces at least one
toxic effect on a tissue or cell, comprising: (a) preparing a gene
expression profile of a tissue or cell sample exposed to said
compound; and (b) comparing the gene expression profile to a
database comprising an adequate amount of the data or information
of Tables 5A-5XX to determine whether the compound induces at least
one toxic effect on the tissue or cell.
2. A method of claim 1, wherein the gene expression profile
prepared from the tissue or cell sample comprises the level of
expression for at least one gene.
3. A method of claim 2, wherein the level of expression is compared
to a Tox Mean and/or Non-tox Mean value in Tables 5A-5XX.
4. A method of claim 3, wherein the level of expression is
normalized prior to comparison.
5. A method of claim 1, wherein the database comprises
substantially all of the data or information in Tables 5A-5XX.
6. A method of predicting at least one toxic effect of a compound,
comprising: (a) detecting the level of expression in a tissue or
cell sample exposed to the compound of two or more genes from
Tables 5A-5XX; wherein differential expression of the genes in
Tables 5A-5XX is indicative of at least one toxic effect.
7. A method of predicting the progression of a toxic effect of a
compound, comprising: (a) detecting the level of expression in a
tissue or cell sample exposed to the compound of two or more genes
from Tables 5A-5XX; wherein differential expression of the genes in
Tables 5A-5XX is indicative of toxicity progression.
8. A method of predicting the hepatotoxicity of a compound,
comprising: (a) detecting the level of expression in a tissue or
cell sample exposed to the compound of two or more genes from
Tables 5A-5XX; wherein differential expression of the genes in
Tables 5A-5XX is indicative of hepatotoxicity.
9. A method of identifying an agent that modulates the onset or
progression of a toxic response, comprising: (a) exposing a cell to
the agent and a known toxin; and (b) detecting the expression level
of two or more genes from Tables 5A-5XX; wherein differential
expression of the genes in Tables 5A-5XX is indicative of
toxicity.
10. (canceled)
11. The method of claim 6, wherein the expression levels of at
least 3 genes are detected.
12. The method of claim 6, wherein the expression levels of at
least 5 genes are detected.
13. The method of claim 6, wherein the expression levels of at
least 10 genes are detected.
14. The method of claim 6, wherein the expression levels of at
least 50 genes are detected.
15. The method of claim 6, wherein the expression levels of at
least 100 genes are detected.
16. The method of claim 6, wherein the expression levels of at
least 500 genes are detected.
17. A method of claim 6, wherein substantially all of the genes in
Tables 5A-5XX are detected.
18. (canceled)
19. A method of claim 6, wherein the compound exposure is in
vitro.
20. A method of claim 19, wherein the cell sample comprises rat
hepatocytes.
21-62. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Applications 60/353,171, filed
Feb. 4, 2002; 60/363,534, filed Mar. 13, 2002; 60/371,135, filed
Apr. 10, 2002; 60/371,134, filed Apr. 10, 2002; 60/370,248, filed
Apr. 8, 2002; 60/371,150, filed Apr. 10, 2002; 60/371,413, filed
Apr. 11, 2002; 60/373,601, filed Apr. 19, 2002; 60/374,139, filed
Apr. 22, 2002; 60/394,253, filed Jul. 9, 2002; 60/378,652, filed
May 9, 2002; 60/373,602, filed Apr. 19, 2002; 60/378,653, filed May
9, 2002; 60/378,665, filed May 9, 2002; 60/378,370, filed May 8,
2002; 60/394,230, filed Jul. 9, 2002; and 60/407,688, filed Sep. 4,
2002, all of which are herein incorporated by reference in their
entirety.
[0002] This application is also related to pending U.S. application
Ser. No. 09/917,800, filed Jul. 31, 2001, 10/060,087, filed Jan.
31, 2002, and PCT/US03/______, entitled "Molecular Hepatotoxicology
Modeling," filed Jan. 31, 2003, as well as to PCT Application
PCT/US01/23872, filed Jul. 31, 2001, all of which are herein
incorporated by reference in their entirety.
SEQUENCE LISTING SUBMISSION ON COMPACT DISC
[0003] The Sequence Listing submitted concurrently herewith on
compact disc under 37 C.F.R. .sctn..sctn.1.821(c) and 1.821(e) is
herein incorporated by reference in its entirety. Three copies of
the Sequence Listing, one on each of three compact discs are
provided. Copy 1 and Copy 2 are identical. Copies 1 and 2 are also
identical to the CRF. Each electronic copy of the Sequence Listing
was created on Feb. 3, 2003 with a file size of 6321 KB. The file
names are as follows: Copy 1--g15113wo.txt; Copy 2--g15113wo.txt;
CRF--g15113wo.txt.
BACKGROUND OF THE INVENTION
[0004] The need for methods of assessing the toxic impact of a
compound, pharmaceutical agent or environmental pollutant on a cell
or living organism has led to the development of procedures which
utilize living organisms as biological monitors. The simplest and
most convenient of these systems utilize unicellular microorganisms
such as yeast and bacteria, since they are most easily maintained
and manipulated. Unicellular screening systems also often use
easily detectable changes in phenotype to monitor the effect of
test compounds on the cell. Unicellular organisms, however, are
inadequate models for estimating the potential effects of many
compounds on complex multicellular animals, as they do not have the
ability to carry out biotransformations to the extent or at levels
found in higher organisms.
[0005] The biotransformation of chemical compounds by multicellular
organisms is a significant factor in determining the overall
toxicity of agents to which they are exposed. Accordingly,
multicellular screening systems or screening systems using isolated
eukaryotic cells may be preferred or required to detect the toxic
effects of compounds. The use of multicellular organisms as
toxicology screening tools has been significantly hampered,
however, by the lack of convenient screening mechanisms or
endpoints, such as those available in yeast or bacterial systems.
In addition, previous attempts to produce toxicology prediction
systems have failed to provide the necessary modeling data and
statistical information to accurately predict toxic responses
(e.g., WO 00/12760, WO 00/47761, WO 00/63435, WO 01/32928, WO
01/38579).
SUMMARY OF THE INVENTION
[0006] The present invention is based on the elucidation of the
global changes in gene expression in primary hepatocytes exposed to
known toxins, in particular hepatotoxins, as compared to unexposed
cells as well as the identification of individual genes that are
differentially expressed upon toxin exposure.
[0007] In various aspects, the invention includes methods of
predicting at least one toxic effect of a compound, predicting the
progression of a toxic effect of a compound, and predicting the
hepatoxicity of a compound. The invention also includes methods of
identifying agents that modulate the onset or progression of a
toxic response. Also provided are methods of predicting the general
pathology classes and cellular pathways that a compound modulates
in a cell. The invention includes methods of identifying agents
that modulate protein activities.
[0008] In a further aspect, the invention provides probes
comprising sequences that specifically hybridize to genes in Tables
1-5XX. Also provided are solid supports comprising at least two of
the previously mentioned probes. The invention also includes a
computer system that has a database containing information
identifying the expression level in a tissue or cell sample exposed
to a hepatotoxin of a set of genes comprising at least two genes in
Tables 1-5XX.
DETAILED DESCRIPTION
[0009] Many biological functions are accomplished by altering the
expression of various genes through transcriptional (e.g. through
control of initiation, provision of RNA precursors, RNA processing,
etc.) and/or translational control. For example, fundamental
biological processes such as cell cycle, cell differentiation and
cell death are often characterized by the variations in the
expression levels of groups of genes.
[0010] Changes in gene expression are also associated with the
effects of various chemicals, drugs, toxins, pharmaceutical agents
and pollutants on an organism or cells. For example, the lack of
sufficient expression of functional tumor suppressor genes and/or
the over expression of oncogene/protooncogenes after exposure to an
agent could lead to tumorgenesis or hyperplastic growth of cells
(Marshall, Cell, 64: 313-326 (1991); Weinberg, Science,
254:1138-1146 (1991)). Thus, changes in the expression levels of
particular genes (e.g. oncogenes or tumor suppressors) may serve as
signposts for the presence and progression of toxicity or other
cellular responses to exposure to a particular compound.
[0011] Monitoring changes in gene expression may also provide
certain advantages during drug screening and development. Often
drugs are screened for the ability to interact with a major target
without regard to other effects the drugs have on cells. These
cellular effects may cause toxicity in the whole animal, which
prevents the development and clinical use of the potential
drug.
[0012] The present inventors have examined primary rat hepatocytes
exposed to the known hepatotoxins which induce detrimental liver
effects, to identify global and individual changes in gene
expression induced by these compounds. These global changes in gene
expression, which can be detected by the production of expression
profiles, as well as the individual genes, provide useful toxicity
markers that can be used to monitor toxicity and/or toxicity
progression by a test compound. Expression profiles, as well as the
individual markers, may also be used to monitor or detect various
disease or physiological states, disease progression, drug efficacy
and drug metabolism.
Identification of Toxicity Markers
[0013] To evaluate and identify gene expression changes that are
predictive of toxicity, studies using selected compounds with well
characterized toxicity have been conducted by the present inventors
to catalogue altered gene expression during exposure in vivo and in
vitro. In the present study, amiodarone,
alpha-naphthylisothiocyante (ANIT), acetaminophen (APAP), AY-25329,
carbamazepine, carbon tetrachloride, chlorpromazine, CI-1000,
clofibrate, CPA, diclofenac, diflunisal, dimethylnitrosamine (DMN),
17.alpha.-ethinylestradiol, gemfibrozil (Lopid.RTM.), hydrazine,
imipramine (Janimine), indomethacin, lipopolysaccharide, lovastatin
(Mevacor.RTM.), methotrexate, phenobarbital, tacrine, tamoxifen,
tetracycline, valproate and Wy-14643 were selected as a known
hepatotoxins.
[0014] Amiodarone (Cordarone.RTM.) is an anti-arrhythmic agent
whose chemical structure contains a benzofuran ring (ring A)
coupled to a p-OH-benzene structure substituted with 2 iodines and
a diethyl-ethanolamine side chain (ring B). This drug is known to
cause damage to the liver and has been shown to adversely effect
the mitochondria by uncoupling oxidative phosphorylation and
inhibiting beta-oxidation and respiration. Inhibition of
respiration decreases ATP and increases production of reactive
oxygen species, which in turn cause lipid peroxidation. The
steatosis and hepatitis observed following treatment with
amiodarone are believed to be due, at least in part, to lipid
peroxidation products (Spaniol et al., J Hepatol 35(5):628-636
(2001); Berson et al., Gastroenterology 114:764-774, (1998)).
[0015] Aromatic and aliphatic isothiocyanates are commonly used
soil fumigants and pesticides (Shaaya et al., Pesticide Science
44(3):249-253 (1995); Cairns et al., J Assoc Official Analytical
Chemists 71(3):547-550 (1988)). These compounds are also
environmental hazards, because they remain as toxic residues in
plants (Cerny et al., J Agricultural and Food Chemistry
44(12):3835-3839 (1996)) and because they are released from the
soil into the surrounding air (Gan et al., J Agricultural and Food
Chemistry 46(3):986-990 (1998)).
[0016] Exposure to .alpha.-naphthylisothiocyanate (ANIT) has been
shown to increase serum levels of total bilirubin, alkaline
phosphatase, serum glutamic oxaloacetic transaminase and serum
glutamic pyruvic transaminase, while total bile flow was reduced,
all of which are indications of severe biliary dysfunction. ANIT
also induces jaundice and cholestatis (the condition caused by
failure to secrete bile, resulting in plasma accumulation of bile
substances, liver cell necrosis and bile duct obstruction) (Tanaka
et al., Clinical and Experimental Pharmacology and Physiology
20:543-547 (1993)). ANIT fails to produce extensive necrosis, but
was found to produce inflammation and edema in the portal tract of
the liver (Maziasa et al., Toxicol Appl Pharmacol 110:365-373
(1991)). ANIT-induced hepatotoxicity may also characterized by
cholangiolitic hepatitis and bile duct damage. Acute hepatotoxicity
caused by ANIT in rats is manifested as neutrophil-dependent
necrosis of bile duct epithelial cells (BDECs) and hepatic
parenchymal cells. These changes mirror the cholangiolitic
hepatitis found in humans (Hill, Toxicol Sci 47:118-125
(1999)).
[0017] Histological changes include an infiltration of
polymorphonuclear neutrophils and elevated number of apoptotic
hepatocytes (Calvo et al., J Cell Biochem 80(4):461-470 (2001)).
Other known hepatotoxic effects of exposure to ANIT include a
damaged antioxidant defense system, decreased activities of
superoxide dismutase and catalase (Ohta et al., Toxicology
139(3):265-275 (1999)), and the release of proteases from the
infiltrated neutrophils, alanine aminotransferase, cathepsin G,
elastase, which mediate hepatocyte killing (Hill et al., Toxicol
Appl Pharmacol 148(1):169-175 (1998)).
[0018] Acetominophen (APAP) is a widely used analgesic and
antipyretic agent that is an effective substitute for aspirin.
Although acetaminophen does not have anti-inflammatory properties,
it is preferably given to patients with ulcers or patients in whom
prolonged clotting times would not be desirable. It also preferably
taken by people who do not tolerate aspirin well.
[0019] Acetominophen is metabolized to N-acetyl-p-benzoquinoneimine
(NAPQI) by N-hydroxylation in a cytochrome P450-mediated process.
This highly reactive intermediate, which reacts with sulfhydryl
groups in glutathione, and in other liver proteins following the
depletion of glutathione, can cause centrilobular hepatic necrosis
(particularly in zone 3), renal tubular necrosis, and hepatic and
renal failure (Goodman and Gilman's The Pharmacological Basis of
Therapeutics. Ninth Ed., Hardman et al., eds., pp. 631-633,
McGraw-Hill, New York, 1996; Chanda et al., Hepatology
21(2):477-486 (1995)). Less serious side effects include skin
rashes (erythemas and urticarias) and allergic reactions.
[0020] Upon treatment of rats with acetaminophen, hepatotoxicity
can be observed 24 hours after dosing, as determined by
statistically significant elevations of ALT and AST in the serum
and by hepatocellular necrosis visualized at the light microscopic
level (Hessel et al., Braz J Med Biol Res 29(6):793-796 (1996);
Bruck et al., Dig Dis Sci 44(6):1228-1235 (1999)). High, but
non-lethal, doses of acetaminophen given to rats also produced
elevated levels of genes involved in hepatic acute phase response
and liver cell maintenance and repair: arginase, beta-fibrinogen,
alpha 1-acid glycoprotein, alpha-tubulin, histone 3, TGF beta and
cyclin d. Expression levels of genes regulated by the cell cycle
were decreased (Tygstrup et al., J Hepatol 25(2):183-190 (1996);
Tygstrup et al., J Hepatol 27(1):156-162 (1997)). In mice,
expression levels of genes that encode growth arrest and cell cycle
regulatory proteins were increased, along with expression levels of
stress-induced genes, transcription factor LRG-21, SOCS-2 (cytokine
signaling repressor) and PAI-1 (plasminogen activator inhibitor-1)
(Reilly et al., Biochem Biophys Res Comm 282(1):321-328
(2001)).
[0021] AY-25329 is a phenothiazine that has been shown to be toxic
in liver and in kidney tissue, where it can cause nephrosis.
Phenothiazines are a class of psychoactive drugs that are used to
treat schizophrenia, paranoia, mania, hyperactivity in children,
some forms of senility, and anxiety
(http://www.encyclopedia.com/articlesnew/36591.html). Side effects
associated with prolonged use of these drugs are reduced blood
pressure, Parkinsonism, reduction of motor activity, and visual
impairment.
[0022] The present inventors have noted indications of liver and
renal effects of AY-25329 by changes in serum chemistry. As early
as 6 hours after the first dose, statistically significant
increases in serum levels of creatinine, BUN, ALT, triglycerides
and cholesterol were observed. Most of these markers of renal and
liver dysfunction remained altered throughout the 14 day study
period. Light microscopic analysis revealed effects in the liver as
early as 6 and 24 hours, as evidenced by an increased number of
hepatocytic mitotic figures and decreased glycogen content.
Following 14 days of repeated dosing, nephrosis and alterations in
the peripheral lobes of the liver and in the cytoplasm of
hepatocytes were evident in rats dosed with 250 mg/kg/day of
AY-25329.
[0023] Carbamazepine (Tegretol.RTM.) is an anti-epieleptic agent.
In rats, it has been shown to induce a number of cytochrome P450
enzymes, in particular CYP2B, and the drug may also cause
steatohepatitis in humans (Tateishi et al., Chem Biol Interact
117:257-268 (1999); Grieco et al., Eur J Gastroenterol
13(8):973-975 (2001)).
[0024] The pathogenesis of acute carbon tetrachloride
(CCl.sub.4)-induced hepatotoxicity follows a well-characterized
course in humans and experimental animals resulting in
centrilobular necrosis and steatosis, followed by hepatic
regeneration and tissue repair. Severity of the hepatocellular
injury is also dose-dependent and may be affected by species, age,
gender and diet.
[0025] Differences in susceptibility to CCl.sub.4 hepatotoxicity
are primarily related to the ability of the animal model to
metabolize CCl.sub.4 to reactive intermediates. CCl.sub.4-induced
hepatotoxicity is dependent on CCl.sub.4 bioactivation to
trichloromethyl free radicals by cytochrome P450 enzymes (CYP2E1),
localized primarily in centrizonal hepatocytes. Formation of the
free radicals leads to membrane lipid peroxidation and protein
denaturation resulting in hepatocellular damage or death.
[0026] The onset of hepatic injury is rapid following acute
administration of CCl.sub.4 to male rats. Morphologic studies have
shown cytoplasmic accumulation of lipids in hepatocytes within 1 to
3 hours of dosing, and by 5 to 6 hours, focal necrosis and hydropic
swelling of hepatocytes are evident. Centrilobular necrosis and
inflammatory infiltration peak by 24 to 48 hours post dose. The
onset of recovery is also evident within this time frame by
increased DNA synthesis and the appearance of mitotic figures.
Removal of necrotic debris begins by 48 hours and is usually
completed by one week, with full restoration of the liver by 14
days.
[0027] Increases in serum transaminase levels also parallel
CCl.sub.4-induced hepatic histopathology. In male Sprague Dawley
(SD) rats, alanine aminotrasferase (ALT) and aspartate
aminotransferase (AST) levels increase within 3 hours of CCl.sub.4
administration (0.1, 1, 2, 3, 4 mL/kg, ip; 2.5 mL/kg, po) and reach
peak levels (approximately 5-10 fold increases) within 48 hours
post dose. Significant increases in serum-glutathione s-transferase
(-GST) levels have also been detected as early as 2 hours after
CCl.sub.4 administration (25 L/kg, po) to male SD rats.
[0028] At the molecular level, induction of the growth-related
proto-oncogenes, c-fos and c-jun, is reportedly the earliest event
detected in an acute model of CCl.sub.4-induced hepatotoxicity
(Schiaffonato et al., Liver 17:183-191 (1997)). Expression of these
early-immediate response genes has been detected within 30 minutes
of a single dose of CCl.sub.4 to mice (0.05-1.5 mL/kg, ip) and by 1
to 2 hours post dose in rats (2 mL/kg, po; 5 mL/kg, po)
(Schiaffonato et al., supra, and Hong et al., Yonsei Medical J
38:167-177 (1997)). Similarly, hepatic c-myc gene expression is
increased by 1 hour following an acute dose of CCl.sub.4 to male SD
rats (5 mL/kg, po) (Hong et al., supra). Expression of these genes
following exposure to CCl.sub.4 is rapid and transient. Peak
hepatic mRNA levels for c-fos, c-jun, and c-myc, after acute
administration of CCl.sub.4 have been reported at 1 to 2 hours, 3
hours, and 1 hour post dose, respectively.
[0029] The expression of tumor necrosis factors (TNF-.alpha.) is
also increased in the livers of rodents exposed to CCl.sub.4, and
TNF-.alpha. has been implicated in initiation of the hepatic repair
process. Pre-treatment with anti-TNF-.alpha. antibodies has been
shown to prevent CCl.sub.4-mediated increases in c-jun and c-fos
gene expression, whereas administration of TNF-.alpha. induced
rapid expression of these genes (Bruccoleri et al., Hepatol
25:133-141 (1997)). Up-regulation of transforming growth
factor-.beta. (TGF-.beta.) and transforming growth factor receptors
(TBRI-III) later in the repair process (24 and 48 hours after
CCl.sub.4 administration) suggests that TGF-.beta. may play a role
in limiting the regenerative response by induction of apoptosis
(Grasl-Kraupp et al., Hepatol 28:717-7126 (1998)).
[0030] Chlorpromazine (Thorazine.RTM.) is a central nervous system
depressant that is used as a sedative and also as an anti-nausea or
anti-itching medication. The mechanism of action is not known. The
drug induces canalicular cholestasis, a condition characterized by
a decrease in the volume of bile formed and impaired secretion of
solutes into bile, resulting in elevated serum levels of bile salts
and bilirubin. Chlorpromazine has also been shown to inhibit bile
acid uptake and canalicular contractility. Bile plugs can form in
the bile ducts and canaliculi. Drug-induced cholestasis is also
associated with cell swelling, inflammation and cell death
(Casarett and Doull's Toxicology: The Basic Science of Poisons, 6th
Ed., Klaassen et al. eds., pp. 476-486, McGraw-Hill Medical Pub.
Div., New York, 2001).
[0031] CI-1000 (4H-pyrrolo:3,2-d:pyrimidin-4-one,
2-amino-3,5-dihydro-7-(3-thienylmethyl)-monohydrochloride
monohydrate) is a compound with anti-inflammatory properties. After
treatment with CI-1000, increased serum ALT levels, a standard
marker of liver toxicity, were observed in dogs.
[0032] Clofibrate, a halogenated phenoxypropanoic acid derivative
(ethyl ester of clofibric acid), is an antilipemic agent. The exact
mechanism by which clofibrate lowers serum concentrations of
triglycerides and low-density lipoprotein (LDL) cholesterol, as
well as raising high-density lipoprotein (HDL) cholesterol is
uncertain. The drug has several antilipidemic actions, including
activating lipoprotein lipase, which enhances the clearance of
triglycerides and very-low-density lipoprotein (VLDL) cholesterol,
inhibition of cholesterol and triglyceride biosynthesis,
mobilization of cholesterol from tissues, increasing fecal
excretion of neutral steroids, decreasing hepatic lipoprotein
synthesis and secretion, and decreasing free fatty acid
release.
[0033] Clofibrate has a number of effects on the rat liver,
including hepatocellular hypertrophy, cellular proliferation,
hepatomegaly, induction of CYP450 isozymes, and induction of
palmitoyl CoA oxidation. Long term administration of clofibrate
causes increased incidence of hepatocellular carcinoma, benign
testicular Leydig cell tumors, and pancreatic acinar adenomas in
rats. Clofibrate induces proliferation of peroxisomes in rodents
and this effect, rather than genotoxic damage, is believed to be
the causative event in rodent carcinogenesis (AHFS Drug Information
Handbook 2001, McEvoy, ed., pp. 1735-1738; Electronic Physicians'
Desk Reference-Atromid-S (clofibrate) at www.pdr.nct; Brown and
Goldstein, "Drugs used in the treatment of hyperliproteinemias," in
Goodman and Gilman's The Pharmacological Basis of Therapeutics.
Eighth ed., Goodman et al., eds., pp. 874-896, Pergamon Press, New
York, 1990).
[0034] Clofibrate also increases hepatic lipid content and alters
its normal composition by significantly increasing levels of
phosphatidylcholine and phosphatidyl-ethanolamine (Adinehzadeh et
al., Chem Res Toxicol 11(5):428-440 (1998)). A rat study of liver
hyperplasia and liver tumors induced by peroxisome proliferators
revealed that administration of clofibrate increased levels of
copper and altered copper-related gene expression in the neoplastic
liver tissues. Down-regulation of the ceruloplasmin gene and of the
Wilson's Disease gene (which encodes P-type ATPase), along with
up-regulation of the metallothionein gene, were noted in these
tissues (Eagon et al., Carcinogenesis 20(6):1091-1096 (1999)).
Clofibrate-induced peroxisome proliferation and carcinogenicity are
believed to be rodent-specific, and have not been demonstrated in
humans.
[0035] Cyproterone acetate (CPA) is a potent androgen antagonist
and has been used to treat acne, male pattern baldness, precocious
puberty, and prostatic hyperplasia and carcinoma (Goodman &
Gilman's The Pharmacological Basis of Therapeutics 9.sup.th ed., p.
1453, J. G. Hardman et al., Eds., McGraw Hill, New York, 1996).
Additionally, CPA has been used clinically in hormone replacement
therapy to protect the endometrium and decrease menopausal symptoms
and the risk of osteoporotic fracture (Schneider, "The role of
antiandrogens in hormone replacement therapy," Climacteric 3
(Suppl. 2): 21-27 (2000)).
[0036] In experiments with rats, CPA was shown to induce
unscheduled DNA synthesis in vitro. After a single oral dose,
continuous DNA repair activity was observed after 16 hours. CPA
also increased the occurrence of S phase cells, which corroborated
the mitogenic potential of CPA in rat liver (Kasper et al.,
Carcinogenesis 17(10): 2271-2274 (1996)). CPA has also been shown
to produce cirrhosis in humans (Garty et al., Eur J Pediatr 158(5):
367-370 (1999)).
[0037] Diclofenac, a non-steroidal anti-inflammatory drug, has been
frequently administered to patients suffering from rheumatoid
arthritis, osteoarthritis, and ankylosing spondylitis. Following
oral administration, diclofenac is rapidly absorbed and then
metabolized in the liver by cytochrome P450 isozyme of the CYC2C
subfamily (Goodman & Gilman's The Pharmacological Basis of
Therapeutics 9.sup.th ed., p. 637, J. G. Hardman et al., eds.,
McGraw Hill, New York, 1996). In addition, diclofenac has been
applied topically to treat pain due to corneal damage (Jayamanne et
al., Eye 11(Pt. 1): 79-83 (1997); Dornic et al., Am J Opthalmol
125(5): 719-721 (1998)).
[0038] Although diclofenac has numerous clinical applications,
adverse side-effects have been associated with the drug, such as
corneal complications, including corneal melts, ulceration, and
severe keratopathy (Guidera et al., Ophthalmology 108(5): 936-944
(2001)). Another study investigated 180 cases of patients who had
reported adverse reactions to diclofenac to the Food and Drug
Administration (Banks et al., Hepatology 22(3): 820-827 (1995)). Of
the 180 reported cases, the most common symptom was jaundice (75%
of the symptomatic patients). Liver sections were taken and
analyzed, and hepatic injury was apparent one month after drug
treatment. An additional report showed that a patient developed
severe hepatitis five weeks after beginning diclofenac treatment
for osteoarthritis (Bhogaraju et al., South Med J 92(7): 711-713
(1999)).
[0039] In one study on diclofenac-treated Wistar rats (Ebong et
al., Afr J Med Sci 27(3-4): 243-246 (1998)), diclofenac treatment
induced an increase in serum chemistry levels of alanine
aminotransferase, aspartate aminotransferase, methaemoglobin, and
total and conjugated bilirubin. Additionally, diclofenac enhanced
the activity of alkaline phosphatase and 5'nucleotidase. A study on
humans revealed elevated levels of hepatic transaminases and serum
creatine when compared to the control group (McKenna et al., Scand
J Rheumatol 30(1): 11-18 (2001)).
[0040] Diflunisal, a non-steroidal anti-inflammatory drug (NSAID),
is a difluorophenyl derivative of salicylic acid (Goodman &
Gilman's The Pharmacological Basis of Therapeutics 9.sup.th ed., p.
631, J. G. Hardman et al., Eds., McGraw Hill, New York, 1996). It
is most frequently used in the treatment of osteoarthritis and
musculoskeletal strains. NSAIDs have analgesic, antipyretic and
anti-inflammatory actions, however, hepatotoxicity is known to be
an adverse side effect of NSAID treatment (Masubuchi et al., J
Pharmacol Exp Ther 287:208-213 (1998)). Diflunisal has been shown
to be less toxic than other NSAIDs, but it can eventually have
deleterious effects on platelet or kidney function (Bergamo et al.,
Am J Nephrol 9:460-463 (1989)). Other side effects that have been
associated with diflunisal treatment are diarrhea, dizziness,
drowsiness, gas or heartburn, headache, nausea, vomiting, and
insomnia
(http://arthritisinsight.com/medical/meds/dolobid.html).
[0041] In a comparative hepatotoxicity study of 18 acidic NSAIDs,
diflunisal was shown to increase LDH leakage in rat hepatocytes, a
marker for cell injury, when compared to control samples.
Additionally, treatment with diflunisal led to decreased
intracellular ATP concentrations. In a study comparing the effects
of diflunisal and ibuprofen, both drugs appeared to cause abdominal
cramping, even during short-term usage. Because the toxic dosages
were selected to be below the level at which gastric ulceration
occurs, more severe gastrointestinal effects were not detected.
But, increased serum levels of creatinine, a sign of renal injury,
were also observed (Muncie et al., Clin Ther 11:539-544
(1989)).
[0042] Another model compound, dimethylnitrosamine (DMN), is a
known carcinogen and inducer of liver fibrosis and lipid
peroxidation. DMN causes oxidative stress in liver cells, which may
be the link between chronic liver damage and liver fibrosis. Rats
treated with DMN showed diffuse fibronectin deposition, elevated
hydroxyproline levels (an indicator of fibrosis), increased levels
of collagens, fibrous septa, and impaired oxidative balance. Serum
levels of ALT and malondialdehyde (MDA) were increased, while serum
levels of SOD were decreased (Vendemiale et al., Toxicol Appl
Pharmacol 175(2):130-139 (2001); Liu et al., Zhonghua Gan Zang Bing
Za Zhi 9 Suppl: 18-20 (2001)). Other studies in rats have noted
severe centrilobular congestion and haemorrhagic necrosis several
days after a three-day period of DMN administration. Following
additional periods of DMN treatment, the rats developed
centrilobular necrosis and intense neutrophilic infiltration, which
progressed to severe centrilobular necrosis, fiber deposition,
focal fatty deposits, bile duct proliferation, bridging necrosis
and fibrosis around the central veins (cirrhosis-like symptoms). A
decrease in total protein and increase in DNA were also observed
(George et al. (2001) Toxicology 156(2-3):129-138).
[0043] 17.alpha.-ethinylestradiol, a synthetic estrogen, is a
component of oral contraceptives, often combined with the
progestational compound norethindrone. It is also used in
post-menopausal estrogen replacement therapy (PDR 47.sup.th Ed.,
pp. 2415-2420, Medical Economics Co., Inc., Montvale, N.J., 1993;
Goodman & Gilman's The Pharmacological Basis of Therapeutics
9.sup.th Ed., pp. 1419-1422, J. G. Hardman et al. Eds., McGraw
Hill, New York, 1996).
[0044] The most frequent adverse effects of
17.alpha.-ethinylestradiol usage are increased risks of
cardiovascular disease: myocardial infarction, thromboembolism,
vascular disease and high blood pressure, and of changes in
carbohydrate metabolism, in particular, glucose intolerance and
impaired insulin secretion. There is also an increased risk of
developing benign hepatic neoplasia. Because this drug decreases
the rate of liver metabolism, it is cleared slowly from the liver,
and carcinogenic effects, such as tumor growth, may result.
[0045] 17.alpha.-ethinylestradiol has been shown to cause a
reversible intrahepatic cholestasis in male rats, mainly by
reducing the bile-salt-independent fraction of bile flow (BSIF)
(Koopen et al., Hepatology 27:537-545 (1998)). Plasma levels of
bilirubin, bile salts, aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) in this study were not changed. This study
also showed that 17.alpha.-ethinylestradiol produced a decrease in
plasma cholesterol and plasma triglyceride levels, but an increase
in the weight of the liver after 3 days of drug administration,
along with a decrease in bile flow. Further results from this study
are as follows. The activities of the liver enzymes leucine
aminopeptidase and alkaline phosphatase initially showed
significant increases, but enzyme levels decreased after 3 days.
Bilirubin output increased, although glutathione (GSH) output
decreased. The increased secretion of bilirubin into the bile
without affecting the plasma level suggests that the increased
bilirubin production must be related to an increased degradation of
heme from heme-containing proteins. Similar results were obtained
in another experiment (Bouchard et al., Liver 13:193-202 (1993)) in
which the livers were also examined by light and electron
microscopy. Daily doses of 17.alpha.-ethinylestradiol have been
shown to cause cholestasis as well, although, following drug
treatment, bile flow rates gradually returned to normal (Hamada et
al., Hepatology 21:1455-1464 (1995)). Liver hyperplasia, possibly
in response to the effects of tumor promoters, has also been
observed (Mayol, Carcinogenesis 13:2381-2388 (1992)).
[0046] The lipid-lowering drug gemfibrozil (Lopid.RTM.) is a know
peroxisome proliferator in liver tissue, causing both hyperplasia
and enlargement of liver cells. Upon exposure to gemfibrozil,
hepatocarcinogenesis has been observed in rats and mice, and a
decrease in alpha-tocopherol and an increase in DT-diaphorase
activity have been observed in rats and hamsters (impaired
antioxidant capability). Peroxisome proliferators increase the
activities of enzymes involved in peroxisomal beta-oxidation and
omega-hydroxylation of fatty acids, which results in oxidative
stress (O'Brien et al., Toxicol Sci 60(2):271-278 (2001); Carthew
et al., J Appl Toxicol 17(1):47-51 (1997)).
[0047] Hydrazine (NH.sub.2.dbd.NH.sub.2), is a component of many
industrial chemicals, such as aerospace and airplane fuels,
corrosion inhibitors, dyes and photographic chemicals. Its
derivatives are used in pharmaceuticals such as hydrazine sulphate,
used to treat cachexia in cancer patients, isoniazid, an
anti-tuberculosis drug, and hydralazine, an anti-hypertensive.
These drugs are metabolized in vivo to produce hydrazine, among
other by-products. Consequently, exposure to hydrazine is by direct
contact, e.g., among military and airline personnel, or the result
of its production in the body, e.g., in patients with cancer or
high blood pressure.
[0048] Studies on rat hepatocytes have shown that hydrazine causes
a dose-dependent loss of viability, leakage of LDH, depletion of
GSH and ATP and a decreased rate of protein synthesis (Delaney et
al., Xenobiotica 25(12):1399-1410 (1995)). When administered to
rats, hepatotoxic changes, characterized by GSH and ATP depletion
and induction of fatty liver (increases in liver weight and
triglycerides, with the appearance of fatty droplets, swelling of
mitochondria and appearance of microbodies) were also found to be
dose-dependent (Jenner et al., Arch Toxicol 68(6):349-357 (1994);
Scales et al., J Toxicol Environ Health 10(6):941-953 (1982)). The
hepatoxicity, as well as renal toxicity, associated with hydrazine
exposure has been linked to free radical damage resulting from
oxidative metabolism by cytochrome P4502E1 (CYP2E1), which
catalyzes the conjugation of free radicals with reduced
glutathione. Although exposure to hydrazine and several hydrazine
derivatives increased enzyme levels in kidney tissue, increased
enzyme levels were not detected in liver tissue (Runge-Morris et
al., Drug Metab Dispos 24(7):734-737 (1996)).
[0049] The mutagenic and hepatocarcinogenic effects of hydrazine
were examined in hamster livers. In vivo, hydrazine reacts with
formaldehyde to form formaldehyde hydrazone
(CH.sub.2.dbd.N--NH.sub.2), an alkylating intermediate that
methylates guanine in DNA. Upon treatment with hydrazine, liver DNA
showed the presence of methylated guanine, DNA adducts and the
impairment of maintenance methylation (impaired methylation of
dcoxycytosine). Hepatic adenomas and carcinomas also developed in a
dose-dependent manner and could be correlated with decreased
maintenance methylation (FitzGerald et al., Carcinogenesis
17(12):2703-2709 (1996)).
[0050] Imipramine, a dibenzazepine derivative, is a tricyclic
anti-depressant agent commonly used for the treatment of major
depression. Experiments in rats have shown that the drug induces
cytochrome P450-mediated formation of reactive metabolites, which
cause acute cell injury. Decreased levels of glutathione and
protein thiols, as well as lactate dehydrogenase leakage, all
standard measures of liver toxicity, were also noted (Masubuchi et
al., Arch Toxicol 73(3):147-151 (1999). On rare occasions,
imipramine has induced cholestasis and hepatitis in humans
(Moskovitz et al., J Clin Psychiatry 43(4):165-066 (1982); Horst et
al., Gastroenterology 79(3):550-544 (1980)).
[0051] Indomethacin is a non-steroidal antiinflammatory,
antipyretic and analgesic drug commonly used to treat rheumatoid
arthritis, osteoarthritis, ankylosing spondylitis, gout and a type
of severe, chronic cluster headache characterized by many daily
occurrences and jabbing pain. This drug acts as a potent inhibitor
of prostaglandin synthesis; it inhibits the cyclooxygenase enzyme
necessary for the conversion of arachidonic acid to prostaglandins
(PDR 47.sup.th Ed., Medical Economics Co., Inc., Montvale, N.J.,
1993; Goodman & Gilman's The Pharmalogical Basis of
Therapeutics 9.sup.th Ed., J. G. Hardman et al. eds., pp.
1074-1075, 1089-1095, McGraw Hill, New York, 1996; Cecil Textbook
of Medicine 20th Ed., part XII, pp. 772-773, 805-808, J. C. Bennett
and F. Plum Eds., W. B. Saunders Co., Philadelphia, 1996).
[0052] The most frequent adverse effects of indomethacin treatment
are gastrointestinal disturbances, usually mild dyspepsia, although
more severe conditions, such as bleeding, ulcers and perforations
can occur. Hepatic involvement is uncommon, although some fatal
cases of hepatitis and jaundice have been reported. Renal toxicity
can also result, particularly after long-term administration. Renal
papillary necrosis has been observed in rats, and interstitial
nephritis with hematuria, proteinuria and nephrotic syndrome have
been reported in humans. Patients suffering from renal dysfunction
risk developing a reduction in renal blood flow, because renal
prostaglandins play an important role in renal perfusion.
[0053] In rats, although indomethacin produces more adverse effects
in the gastrointestinal tract than in the liver, it has been shown
to induce changes in hepatocytic cytochrome P450 In one study, no
widespread changes in the liver were observed, but a mild, focal,
centrilobular response was noted. Serum levels of albumin and total
protein were significantly reduced, while the serum level of urea
was increased. No changes in creatinine or aspartate
aminotransferase (AST) levels were observed (Falzon et al., Br J
exp Path 66:527-534 (1985)). In another rat study, a single dose of
indomethacin was shown to reduce liver and renal microsomal
enzymes, including CYP450, and cause lesions in the GI tract
(Fracasso et al., Agents Actions 31:313-316 (1990)).
[0054] LPS (lipopolysaccharide) is an endotoxin released by
gram-negative bacteria upon breakage or rupture of the cells that
induces an acute inflammatory response in mammals and that can
cause septic shock. LPS is also a research tool used to initiate
liver injury in rats through an inflammatory mechanism. Typically,
the membrane components of LPS are lipid-A, KDO
(2-keto-3-deoxy-octulosonic acid), core polysaccharides and
O-antigen polysaccharides, the polysaccharide units differing from
one bacterium to another (Zinsser Microbiology 20th Ed., Joklik et
al., eds., pp. 82-87, Appleton & Lange, Norwalk, Conn.,
1992).
[0055] Primary rat hepatocytes derived from liver parenchymal cells
and sinusoidal cells of rats that have been exposed to LPS in vivo
can directly respond to LPS in cell culture. Numerous effects of
LPS-induced endotoxemia can be detected, including elevated levels
of nitric oxide synthetase (NOS) with increased nitric oxide and
nitritc production, cellular hypertrophy, vacuolization,
chromosomal emargination, cytoplasmic DNA fragmentation and
necrosis (Pittner et al., Biochem Biophys Res Commun 185(1):430-435
(1992); Laskin et al., Hepatology 22(1):223-234 (1995); Wang et
al., Am J Physiol 269(2 Pt 1):G297-304 (1995)). Other studies have
indicated that the presence of Kupffer cells with primary rat
hepatocytes is essential for the induction of hepatocyte apoptosis
by LPS (Hamada et al., J Hepatol 30(5):807-818 (1999)).
[0056] Exposure of rats or primary hepatocytes to LPS induces the
expression of a number of acute-phase proteins in the liver. Recent
evidence has indicated that rat hepatocytes express soluble CD14
protein, and LPS is capable of markedly increasing levels of CD 14
at both the gene expression and protein expression levels (Liu et
al., Infect Immun 66(11):5089-5098 (1998)). Soluble CD14 is
believed to be a critical LPS recognition protein required for the
activation of a variety of cells to toxic levels of LPS, even in
endothelial and epithelial cells (Pugin et al., Proc Natl Acad Sci
USA 90(7):2744-2748 (1993)). Another key component of the LPS
recognition system is lipopolysaccharide-binding protein (LBP),
which binds to LPS. The LPS-LBP complex interacts with the CD14
receptor, inducing LPS sensitive genes. LBP can be induced in
hepatocytes isolated from rats that have not been primed with LPS,
indicating that this key regulatory pathway is intact in primary
rat hepatocytes (Wan et al., Infect Immun 63(7):2435-2442
(1995)).
[0057] Lovastatin (Mevacor.RTM.) is a cholesterol-lowering agent
belonging to a class of compounds, the statins, that are potent
inhibitors of HMG-CoA reductase. This enzyme catalyzes the
conversion of HMG-CoA to mevalonate, the rate-controlling enzyme in
cholesterol biosynthesis. HMG-CoA reductase inhibitors block the
production of cholesterol in the liver leading to a reduction of
LDL particles in the plasma. Lovastatin has additional effects on
lipid metabolism, including depletion of intracellular pools of
sterols and increased synthesis of LDL receptors, leading to
enhanced removal of LDL and LDL precursors from plasma. Upon
treatment with lovastatin, plasma levels of VLDL, IDL and
triglycerides also decrease. Oral doses of lovastatin are
extensively absorbed by the liver, and the drug is excreted
primarily via the liver; less than 13% of its metabolites are
excreted in the urine (Goodman and Gilman's The Pharmacological
Basis of Therapeutics. Ninth Ed., Hardman et al., eds., pp.
884-888, McGraw-Hill, New York, 1996).
[0058] The most frequent side effects are liver damage,
characterized by elevated levels of hepatic transaminases (e.g.,
AST and ALT), creatinine phosphokinase and alkaline phosphatase,
and myopathy, characterized by muscle pain and destruction of
skeletal muscle cells. Cases of drug-induced hepatitis, accompanied
by jaundice and elevated levels of liver enzymes, have also been
reported, although the symptoms were reversible following
withdrawal of the drug (Huchzermeyer et al., Deutsch Med Wochenschr
120(8):252-256 (1995); Heuer et al., Med Klin 95(11):642-644
(2000)). Histologic examination of affected liver tissue showed
centrilobular necrosis, centrilobular cholestasis, and infiltrates
with mononuclear and polymorphonuclear cells, including eosinophils
(Grimbert et al., Dig Dis Sci 39(9):2032-2033 (1994)).
[0059] Experiments by the present inventors have found that when
rats were dosed with lovastatin, at 9 or 90 mg/kg twice a day, no
effects were noted in liver tissue after 6 or after 24 hours. After
14 days of treatment at the higher dosage, however, liver cells
showed abnormal vacuolization of the cytoplasm. Hepatoxicity data
from other studies of laboratory animals treated with lovastatin
have not been widely reported. Therefore, in order to establish a
more sensitive model for examining the changes in liver tissue
caused by lovastatin, as well as to have a means of examining
changes in expression level of individual genes as a result of
exposure to lovastatin, experiments in cultured hepatocytes were
undertaken.
[0060] Methotrexate is a widely used antineoplastic drug that is
also frequently prescribed for the treatment of psoriasis (a
disease characterized by abnormal proliferation of epidermal
cells), juvenile lymphoblastic leukemia, rheumatoid arthritis, and
a number of other cancerous diseases, such as leukemic meningitis
and choriocarcinoma. Although generally not metabolized, at high
dosages, metabolites such as 7-hydroxy-methotrexate, a nephrotoxin,
do accumulate. Methotrexate polyglutamates are retained in the
kidneys for weeks and in the liver for months ((Goodman and
Gilman's The Pharmacological Basis of Therapeutics. Ninth Ed.,
Hardman et al., eds., pp. 1243-1247, McGraw-Hill, New York,
1996).
[0061] Methotrexate acts to prevent DNA synthesis and cell
replication by inhibiting the rate-limiting enzyme in purine and
thymidine synthesis, dihydrofolate reductase (DHFR) (Goodman and
Gilman's, supra; Schwartz et al., Proc Nat Acad Sci USA
89(2):594-598 (1992)). It also acts as an suppressant of
cell-mediated immune responses. The biochemical toxicology of
methotrexate has been well characterized in man, where long-term
administration produces hepatic fibrosis or cirrhosis, especially
in heavy drinkers, which is linked to persistent, mild-to-moderate,
increases in serum transaminases, alkaline phosphatases and
bilirubin (Reynolds et al., South Med J79(5):536-539 (1986); Tolman
et al., J Rheumatol 12 (Suppl 12):29-34 (1985)). Methotrexate is a
rather long-acting, rapidly reversible DHFR inhibitor, despite its
high affinity for the target enzymes in many cell types, which may
be due to the formation of methotrexate polyglutamates that reduce
the ability of DHFR to pass through cell membranes. The toxic
effects of methotrexate may be due to the depletion of
tetrahydrofolate cofactors that are required for purine and
thymidylate synthesis (methylation reactions in hepatic 1-carbon
metabolism) and to the inhibition of folate-dependent enzymes
involved in the metabolism of purines and thymidylate, the
inhibition caused by the accumulation of methotrexate
polyglutamates and dihydrofolate polyglutamates.
[0062] The mechanism of methotrexate-induced hepatotoxicity is not
yet fully elucidated, partly because the pathological changes in
humans are rather difficult to reproduce in animal models, although
experiments in rats have shown that, in a dose-dependent fashion,
methotrexate produces liver damage ranging from focal to confluent
necrosis of zone 3 hepatocytes, with early stage fibrosis (Hall et
al., Hepatology 14(5):906-10 (1991)). Other studies in rats have
demonstrated that treatment with methotrexate produces
intrahepatocytic fat deposits, along with fatty accumulations in
hepatocytes that range from fine droplets to large vacuoles. The
areas of necrosis showed signs of the hypoxia associated with
congestive failure, as well as anemic infarcts, fibrotic foci of
the collapse type, atrophy of the hepatic cords, and hemosiderosis
(Custer et al., J Natl Cancer Inst 58(4):1011-1015 (1977)).
Hepatotoxicity probably involves interference with triglyceride and
other lipid biosynthetic pathways in the liver. For example,
studies on rats have shown that methotrexate inhibits the
biosynthesis of lipotropic substances such as methionine and
choline through its interference with hepatic 1-carbon metabolism.
The drug also inhibits the activity of vitamin B12, another
lipotropic factor (Tuma et al., Biochem Pharmacol 24:1327-1331
(1975) and impairs RNA and protein synthesis, triglyceride
secretion and total triglyceride esterification (Deboyser et al.,
Toxic in Vitro 6(2):129-132 (1992).
[0063] Methotrexate does not appear to be cytotoxic to cultured
primary hepatocytes following short-term exposures (up to 3.5 hr),
but significant effects on HepG2 growth curves have been observed
at low concentrations during the course of 7-day exposures (Wu et
al., Proc Natl Acad Sci USA 80(10):3078-3080 (1983)). Additionally,
it has been demonstrated that methotrexate increases hepatic
glycogenolysis, oxygen consumption and calcium efflux and decreases
glutathione levels (Yamamoto et al., Biochem Pharmacol
44(4):761-767, (1992); de Oliveira et al., Res Commun Chem Pathol
Pharmacol 53(2):173-181 (1986); Lindenthal et al., Eur J Pharmacol
228(5-6):289-298 (1993)). Experiments on cultured rat hepatocytes
have shown that methotrexate also inhibits the activity of hepatic
N-acetyltransferase 2 (NAT2), although the drug has only a slight
inhibitory effect on rat NAT1, enzymes that catalyze the
acetylation of a variety of therapeutic arylamines (Zaher et al.,
Toxicol in Vitro 11:271-283 (1997)).
[0064] Phenobarbital, a barbiturate, is used as an anti-epileptic,
sedative or hypnotic drug and can also be used to treat neuroses
with related tension states, such as hypertension, coronary artery
disease, gastrointestinal disturbances and preoperative
apprehension. Phenobarbital is also found in medications to treat
insomnia and headaches (Remington: The Science and Practice of
Pharmacy, 19th Ed., A. R. Gennaro ed., pp. 1164-1165, Mack
Publishing Co., Easton, Pa., 1995).
[0065] Phenobarbital induces a variety of drug metabolizing enzymes
such as cytochrome P450 oxidoreductase, aldehyde dehydrogenases,
UDP-glucuronyltransferase, GSTs, epoxide hydrolase, and an
assortment of cytochrome P450 monooxygenases (Waxman et al.,
Biochem J 1281(Pt 3):577-592 (1992); Kaplowitz et al., Biochem J
146(2):351-356 (1975); Tank et al., Biochem Pharmacol
35(24):4563-4569 (1986). The induction of liver enzymes is usually
accompanied by liver enlargement, proliferation of smooth
endoplasmic reticulum, and tumor promotion (Waxman et al., supra;
Rice et al., Carcinogenesis 15(2):395-402 (1994); Nims et al.,
Carcinogenesis 8(1):67-71, (1987). Incidences of cholestasis and
hepatocellular injury have also been found (Selim et al.,
Hepatology 29(5):1347-1351 (1999); Gut et al., Environ Health
Perspect 104(Suppl 6):1211-1218 (1999)). Phenobarbital has been
classified as nongenotoxic hepatocarcinogen as it induces liver
tumors in rodents but lacks detectable signs of genotoxicity using
short term in vivo or in vitro assays (Whysner et al., Pharmacol
Ther 71(1-2):153-191 (1996)).
[0066] The effects of phenobarbital on phospholipid metabolism in
rat liver have been studied. In one study, phenobarbital,
administered intraperitonially (i.p.), was found to cause an
increase in the microsomal phosphatidylcholine content.
Additionally, levels of glycerophosphate acyltransferase (GAT),
phosphatidate cytidylyltransferase (PCT), phosphatidate
phosphohydrolase (PPH) and choline phosphotransferase (CPT) were
significantly increased (Hoshi et al., Chem Pharm Bull 38:3446-3448
(1990)).
[0067] Tacrine (1,2,3,4-tetrahydro-9-aminoacridine-hydrochloride),
a strong acetylcholinesterase (AChE) inhibitor, is used in the
treatment of mild to moderate cases Alzheimer's dimentias.
Alzheimer's patients have synaptic loss, neuronal atrophy and
degeneration of cholinergic nuclei in the forebrain, which are
associated with reduced oxidative metabolism of glucose and
decreased levels of ATP and acetyl CoA. Administration of ACHE
inhibitors, such as tacrine, is designed to increase cholinergic
activity to combat this loss (Weinstock, Neurodegeneration
4(4):349-356 (1995)). The effect seen in the patients is a reversal
of the cognitive and functional decline, but the drug does not
appear to change the neurodegenerative process (Goodman &
Gilman's The Pharmacological Basis of Therapeutics 9.sup.th Ed.,
Hardman et al. eds., p. 174, McGraw Hill, New York, 1996).
[0068] Hepatotoxicty caused by tacrine is typically reversible,
although cases of severe hepatotoxicity have been seen (Blackard et
al., J Clin Gastroenterol 26:57-59 (1998)). The toxicity is
characterized by decreased levels of protein synthesis and the
release of lactate dehydrogenase, as well as by increased
transaminase levels and decreased levels of ATP, glycogen and
glutathione. The decrease in protein synthesis may represent a
signal leading to cell death (Lagadic-Gossmann et al., Cell Biol
Toxicol 14(5):361-373 (1998)).
[0069] Preclinical studies have failed to detect adverse hepatic
events, although tacrine displayed cytotoxicity to human hepatoma
cell lines and primary rat hepatocytes (Viau et al., Drug Chem
Toxicol 16:227-239 (1993)). While hepatotoxicity has been found in
humans, in vivo rat studies have not shown a correlation between
tacrine exposure and hepatotoxicity, and the mechanism of action is
not completely understood. An in vitro study comparing the reaction
of human and rat liver microsomal preparations to tacrine showed
that the two species reacted differently to the drug, suggesting
that the rat may not be the best model for monitoring
tacrine-induced elevations in liver marker enzymes (Woolf et al.,
Drug Metab Dispos 21:874-882 (1993)).
[0070] While tacrine does not reveal classic signs of
hepatotoxicity in rats, gene expression changes due to tacrine
administration can be used to predict that the drug will be a liver
toxin in humans. This suggests that toxicogenomics might be able to
detect drugs that prove to be toxic in the clinic even when
classical but more crude measures in preclinical screening fail to
detect toxicity.
[0071] Tamoxifen is a nonsteroidal anti-estrogen used for breast
cancer in males and females. Tamoxifen has been associated with
changes in liver enzyme levels, disruption of mitochondrial
metabolism and, occasionally, with a spectrum of more severe liver
abnormalities including fatty liver, cholestasis, hepatic necrosis
and NASH (nonalcoholic steatohepatitis) (Duthie et al., Xenobiotica
25(10):1151-1164 (1995); Cardoso et al., Toxicol Appl Pharmacol
176(3):145-152 (2001); Pinol et al., Gastroenterol Hepatol
23(2):57-61 (2000); and Farrell, Semin Liver Dis 22(2):185-194
(2002)). A few of these serious cases included fatalities. A few
cases of liver cancer have also been reported. Additionally,
studies in mice and rats have shown that tamoxifen significantly
alters the activities of liver enzymes and can induce hepatic
carcinomas (Caballero et al., Int J Biochem Cell Biol 33(7):681-690
(2001); Guzelian, Semin Oncol 24(1 Suppl 1):S1-105-121 (1997)).
[0072] Tetracycline is a broad spectrum antibiotic whose main
mechanism of action is the inhibition of bacterial protein
synthesis. Hepatic toxicity, principally steatosis, has been
observed in patients receiving high doses of tetracycline. In rats
and dogs, exposure to tetracycline has been shown to increase
levels of total lipids and triglycerides in liver cells due to
inhibition of mitochondrial lipid metabolism and beta-oxidation
(Lewis et al., Am J Dig Dis 12:429-438, (1967); Amacher et al.,
Fundam Appl Toxicol 40(2):256-263 (1997).
[0073] Valproate (n-dipropylacetic acid, Depakene.RTM.) is
routinely used to treat several types of epileptic seizures-absence
seizures, myoclonic seizures and tonic-clonic seizures. Most other
anti-epileptics are effective against only one type. Valproate acts
on neurons to inhibit the sustained repetitive firing caused by
depolarization of cortical or spinal cord neurons, and a prolonged
recovery of inactivated voltage-activated Na.sup.+ channels
follows. The drug also acts by reducing the low-threshold Ca.sup.2+
current and its multiple mechanisms contribute to its use in
multiple types of seizures. Although valproate does not affect
neuronal responses to GABA, it does increase the activity of the
GABA synthetic enzyme, glutamic acid decarboxylase, and it inhibits
enzymes that degrade GABA, GABA transaminase and succinic
semialdehyde dehydrogenase (Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.,
eds., pp. 462, 476 and 477, McGraw-Hill, New York, 1996).
[0074] The most common side effects are gastrointestinal symptoms,
including anorexia, nausea and vomiting. Effects on the CNS include
sedation, ataxia and tremor. Rash, hair loss, increased appetite
and teratogenic effects have also been observed (Briggs et al., A
Reference Guide to Fetal and Neonatal Risk. Drugs in Pregnancy and
Lactation, 4th ed., p. 869, Williams & Wilkins, Baltimore,
1994). With respect to liver toxicity, valproate produces elevated
levels of hepatic enzymes in about 40% of patients, which may be an
asymptomatic condition, and elevated levels of hepatic lipids.
Fulminant hepatitis, microvesicular steatosis (fatty degeneration),
hepatocyte necrosis and hepatic failure can also result. It is
believed that hepatoxicity is caused by an accumulation of
unsaturated metabolites of valproate, in particular 4-en-valproate,
which is structurally similar to two known hepatotoxins,
4-en-pentanoate and methylenecyclopropylacetic acid (Eadie et al.,
Med Toxicol Adverse Drug Exp 3(2):85-106 (1988)).
[0075] In a study on rats, microvesicular steatosis caused by
valproate was found to be accompanied by myeloid bodies, lipid
vacuoles and mitochondrial abnormalities (Kesterson et al.,
Hepatology 4(6):1143-1152 (1984)). Experiments on cultured rat
hepatocytes have shown that valproate produces a dose-dependent
leakage of lactic acid dehydrogenase and increased amounts of
acyl-CoA esters, compounds that interfere with the beta-oxidation
of fatty acids (Vance et al., Epilepsia 35(5): 1016-1022 (1994)).
Administration of valproate to rats has also been shown to cause
enhanced excretion of dicarboxylic acids, a sign of impaired
mitochondrial beta-oxidation. Other metabolic effects include
hypoglycemia, hyperammonemia, decreased levels of
beta-hydroxybutyrate and carnitine and decreased activities of
acyl-CoA dehydrogenases, enzymes involved in fatty acid oxidation.
mRNA levels of genes involved in fatty acid oxidation, however,
such as the short-, medium- and long-chain acyl-CoA dehydrogenases,
were found to have increased (Kibayashi et al., Pediatr Int
41(1):52-60 (1999)).
[0076] Wy-14643, a tumor-inducing compound that acts in the liver,
has been used to study the genetic profile of cells during the
various stages of carcinogenic development, with a view toward
developing strategies for detecting, diagnosing and treating
cancers (Rockett et al., Toxicology 144(1-3):13-29 (2000)). In
contrast to other carcinogens, Wy-14643 does not mutate DNA
directly. Instead, it acts on the peroxisome proliferator activated
receptor-alpha (PPARalpha), as well as on other signaling pathways
that regulate growth (Johnson et al., J Steroid Biochem Mol Biol
77(1):59-71 (2001)). The effect is elevated and sustained cell
replication, accompanied by a decrease in apoptosis (Rusyn et al.,
Carcinogenesis 21(12):2141-2145 (2000)). These authors (Rusyn et
al.) noted an increase in the expression of enzymes that repair DNA
by base excision, but no increased expression of enzymes that do
not repair oxidative damage to DNA. In a study on rodents, Johnson
et al. noted that Wy-14643 inhibited liver-X-receptor-mediated
transcription in a dose-dependent manner, as well as de novo sterol
synthesis.
[0077] In experiments with mouse liver cells (Peters et al.,
Carcinogenesis 19(11): 1989-1994 (1998)), exposure to Wy-14643
produced increased levels of acyl CoA oxidase and proteins involved
in cell proliferation: CDK-1, 2 and 4, PCNA and c-myc. Elevated
levels may be caused by accelerated transcription that is mediated
directly or indirectly by PPARalpha. It is likely that the
carcinogenic properties of peroxisome proliferators are due to the
PPARalpha-dependent changes in levels of cell cycle regulatory
proteins.
[0078] Another study on rodents (Keller et al., Biochim Biophys
Acta 1102(2):237-244 (1992)) showed that Wy-14643 was capable of
uncoupling oxidative phosphorylation in rat liver mitochondria.
Rates of urea synthesis from ammonia and bile flow, two
energy-dependent processes, were reduced, indicating that the
energy supply for these processes was disrupted as a result of
cellular exposure to the toxin. Wy-14643 has also been shown to
activate nuclear factor kappaB, NADPH oxidase and superoxide
production in Kupffer cells (Rusyn et al., Cancer Res
60(17):4798-4803 (2000)). NADPH oxidase is known to induce
mitogens, which cause proliferation of liver cells.
Toxicity Identification, Prediction and Modeling
[0079] The genes and gene expression information, as well as the
portfolios and subsets of the genes provided in Tables 1-5XX may be
used to predict or identify at least one toxic effect, including
the hepatotoxicity of a test or unknown compound As used, herein,
at least one toxic effect includes, but is not limited to, a
detrimental change in the physiological status of a cell or
organism. The response may be, but is not required to be,
associated with a particular pathology, such as tissue necrosis.
Accordingly, the toxic effect includes effects at the molecular and
cellular level. Hepatotoxicity is an effect as used herein and
includes, but is not limited to, genotoxic and non-genotoxic
carcinogenesis, cholestasis, hepatitis, liver enlargement,
inflammation, necrosis, necrosis with steatosis, peroxisome
proliferation, steatosis, and steatosis with hepatitis. In
addition, hepatoxicity includes the effect of direct-acting agents
(such as metformin, rosiglitazone and dexamethasone), which are
pharmaceuticals that act in the liver, but are not considered toxic
to the liver. Exposure to these agents results in altered gene
expression profiles. As used herein, a gene expression profile
comprises any quantitative representation of the expression of at
least one mRNA species in a cell sample or population and includes
profiles made by various methods such as differential display, PCR,
hybridization analysis, etc.
[0080] In general, assays to predict the toxicity or hepatotoxicity
of a test agent (or compound or multi-component composition)
comprise the steps of exposing a cell population to the test
compound, assaying or measuring the level of relative or absolute
gene expression of one or more of the genes in Tables 5A-5XX and
comparing the identified expression level(s) to the expression
levels disclosed in the Tables and database(s) disclosed herein.
Assays may include the measurement of the expression levels of
about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 75, 100, 200,
300, 400, 500, 1000 or more genes from Tables 5A-5XX to create
multi-gene expression profiles. In some embodiments, all or
substantially all of the genes of Tables 5A-5XX may be used to
predict toxicity, etc. In other embodiments, the genes or subsets
of the genes for each individual toxin model, for instance, the
genes of Table 5A, may be used. An "adequate amount" of the data of
Tables 5A-5XX refers to an amount of information that allows
toxicity identification or prediction (typically 2 or more genes).
"Substantially" or nearly all of the data in the tables refers to
at least about 80% of the data for an individual model.
[0081] In the methods of the invention, the gene expression level
for a gene or genes induced by the test agent, compound or
compositions may be comparable to the levels found in the Tables or
databases disclosed herein if the expression level varies within a
factor of about 2, about 1.5 or about 1.0 fold. In some cases, the
expression levels are comparable if the agent induces a change in
the expression of a gene in the same direction (e.g., up or down)
as a reference toxin. "Comparing" may comprise determining the
relationship of the database information to the sample gene
expression profile with or without application of an algorithm to
the results, differences or similarities between the two.
[0082] The cell population that is exposed to the test agent,
compound or composition may be exposed in vitro or in vivo. For
instance, cultured or freshly isolated hepatocytes, in particular
rat hepatocytes, may be exposed to the agent under standard
laboratory and cell culture conditions. In another assay format, in
vivo exposure may be accomplished by administration of the agent to
a living animal, for instance a laboratory rat.
[0083] Procedures for designing and conducting toxicity tests in in
vitro and in vivo systems are well known, and are described in many
texts on the subject, such as Loomis et al., Loomis's Esstentials
of Toxicology. 4th Ed., Academic Press, New York, 1996; Echobichon,
The Basics of Toxicity Testing, CRC Press, Boca Raton, 1992;
Frazier, editor, In Vitro Toxicity Testing, Marcel Dekker, New
York, 1992; and the like.
[0084] In in vitro toxicity testing, two groups of test organisms
are usually employed: One group serves as a control and the other
group receives the test compound in a single dose (for acute
toxicity tests) or a regimen of doses (for prolonged or chronic
toxicity tests). Because, in some cases, the extraction of tissue
as called for in the methods of the invention requires sacrificing
the test animal, both the control group and the group receiving
compound must be large enough to permit removal of animals for
sampling tissues, if it is desired to observe the dynamics of gene
expression through the duration of an experiment.
[0085] In setting up a toxicity study, extensive guidance is
provided in the literature for selecting the appropriate test
organism for the compound being tested, route of administration.
dose ranges, and the like. Water or physiological saline (0.9% NaCl
in water) is the solute of choice for the test compound since these
solvents permit administration by a variety of routes. When this is
not possible because of solubility limitations, vegetable oils such
as corn oil or organic solvents such as propylene glycol may be
used.
[0086] Regardless of the route of administration, the volume
required to administer a given dose is limited by the size of the
animal that is used. It is desirable to keep the volume of each
dose uniform within and between groups of animals. When rats or
mice are used, the volume administered by the oral route generally
should not exceed about 0.005 ml per gram of animal. Even when
aqueous or physiological saline solutions are used for parenteral
injection the volumes that are tolerated are limited, although such
solutions are ordinarily thought of as being innocuous. The
intravenous LD.sub.50 of distilled water in the mouse is
approximately 0.044 ml per gram and that of isotonic saline is
0.068 ml per gram of mouse. In some instances, the route of
administration to the test animal should be the same as, or as
similar as possible to, the route of administration of the compound
to man for therapeutic purposes.
[0087] When a compound is to be administered by inhalation, special
techniques for generating test atmospheres are necessary. The
methods usually involve aerosolization or nebulization of fluids
containing the compound. If the agent to be tested is a fluid that
has an appreciable vapor pressure, it may be administered by
passing air through the solution under controlled temperature
conditions. Under these conditions, dose is estimated from the
volume of air inhaled per unit time, the temperature of the
solution, and the vapor pressure of the agent involved. Gases are
metered from reservoirs. When particles of a solution are to be
administered, unless the particle size is less than about 2 .mu.m
the particles will not reach the terminal alveolar sacs in the
lungs. A variety of apparatuses and chambers are available to
perform studies for detecting effects of irritant or other toxic
endpoints when they are administered by inhalation. The preferred
method of administering an agent to animals is via the oral route,
either by intubation or by incorporating the agent in the feed.
[0088] When the agent is exposed to cells in vitro or in cell
culture, the cell population to be exposed to the agent may be
divided into two or more subpopulations, for instance, by dividing
the population into two or more identical aliquots. In some
preferred embodiments of the methods of the invention, the cells to
be exposed to the agent are derived from liver tissue. For
instance, cultured or freshly isolated rat hepatocytes may be
used.
[0089] The methods of the invention may be used generally to
predict at least one toxic response, and, as described in the
Examples, may be used to predict the likelihood that a compound or
test agent will induce various specific liver pathologies, such as
genotoxic and non-genotoxic carcinogenesis, cholestasis, direct
action toxicity, hepatitis, liver enlargement, inflammation,
necrosis, necrosis with steatosis, peroxisome proliferation,
steatosis, steatosis with hepatitis, or other pathologies
associated with at least one of the toxins herein described. The
methods of the invention may also be used to determine the
similarity of a toxic response to one or more individual compounds.
In addition, the methods of the invention may be used to predict or
elucidate the potential cellular pathways influenced, induced or
modulated by the compound or test agent due to the similarity of
the expression profile compared to the profile induced by a known
toxin (see Tables 5A-5G, 5J, 5K, 5M-5S, 5U-5Y, 5AA-5EE, 5HH-5JJ,
5MM, 5OO, 5PP and 5SS-5XX). Further, the link between a specific
liver pathology that is the result of exposure to a toxin and a
specific gene expression profile allows for distinction of the
genes in Tables 5A-5XX as markers of liver toxicity.
Diagnostic Uses for the Toxicity Markers
[0090] As described above, the genes and gene expression
information or portfolios of the genes with their expression
information as provided in Tables 5A-5XX may be used as diagnostic
markers for the prediction or identification of the physiological
state of tissue or cell sample that has been exposed to a compound
or to identify or predict the toxic effects of a compound or agent.
For instance, a tissue sample such as a sample of peripheral blood
cells or some other easily obtainable tissue sample may be assayed
by any of the methods described above, and the expression levels
from a gene or genes from Tables 5A-5XX may be compared to the
expression levels found in tissues or cells exposed to the toxins
described herein. These methods may result in the diagnosis of a
physiological state in the cell or may be used to identify the
potential toxicity of a compound, for instance a new or unknown
compound or agent. The comparison of expression data, as well as
available sequence or other information may be done by researcher
or diagnostician or may be done with the aid of a computer and
databases as described below.
[0091] In another format, the levels of a gene(s) of Tables 5A-5XX,
its encoded protein(s), or any metabolite produced by the encoded
protein may be monitored or detected in a sample, such as a bodily
tissue or fluid sample to identify or diagnose a physiological
state of an organism. Such samples may include any tissue or fluid
sample, including urine, blood and easily obtainable cells such as
peripheral lymphocytes.
Use of the Markers for Monitoring Toxicity Progression
[0092] As described above, the genes and gene expression
information provided in Tables 5A-5XX may also be used as markers
for the monitoring of toxicity progression, such as that found
after initial exposure to a drug, drug candidate, toxin, pollutant,
etc. For instance, a tissue or cell sample may be assayed by any of
the methods described above, and the expression levels from a gene
or genes from Tables 5A-5XX may be compared to the expression
levels found in tissue or cells exposed to the hepatotoxins
described herein. The comparison of the expression data, as well as
available sequence or other information may be done by researcher
or diagnostician or may be done with the aid of a computer and
databases.
Use of the Toxicity Markers for Drug Screening
[0093] According to the present invention, the genes identified in
Tables 5A-5XX may be used as markers or drug targets to evaluate
the effects of a candidate drug, chemical compound or other agent
on a cell or tissue sample. The genes may also be used as drug
targets to screen for agents that modulate their expression and/or
activity. In various formats, a candidate drug or agent can be
screened for the ability to simulate the transcription or
expression of a given marker or markers or to down-regulate or
counteract the transcription or expression of a marker or markers.
According to the present invention, one can also compare the
specificity of a drug's effects by looking at the number of markers
which the drug induces and comparing them. More specific drugs will
have less transcriptional targets. Similar sets of markers
identified for two drugs may indicate a similarity of effects. As
used herein, an agent is said to modulate the expression of a
nucleic acid of the invention if it is capable of up- or
down-regulating expression of the nucleic acid in a cell.
[0094] Assays to monitor the expression of a marker or markers as
defined in Tables 5A-5XX may utilize any available means of
monitoring for changes in the expression level of the nucleic acids
of the invention.
[0095] In one assay format, microarrays containing probes to one,
two or more genes from Tables 5A-5XX may be used to directly
monitor or detect changes in gene expression in the treated or
exposed cell. Cell lines, tissues or other samples are first
exposed to a test agent and in some instances, a known toxin, and
the detected expression levels of one or more, or preferably 2 or
more of the genes of Tables 5A-5XX are compared to the expression
levels of those same genes exposed to a known toxin alone.
Compounds that modulate the expression patterns of the known
toxin(s) would be expected to modulate potential toxic
physiological effects in vivo. The genes in Tables 5A-5XX are
particularly appropriate marks in these assays as they are
differentially expressed in cells upon exposure to a known
hepatotoxin.
[0096] In another format, cell lines that contain reporter gene
fusions between the open reading frame and/or the transcriptional
regulatory regions of a gene in Tables 5A-5XX and any assayable
fusion partner may be prepared. Numerous assayable fusion partners
are known and readily available including the firefly luciferase
gene and the gene encoding chloramphenicol acetyltransferase (Alam
et al., Anal Biochem 188:245-254 (1990)). Cell lines containing the
reporter gene fusions are then exposed to the agent to be tested
under appropriate conditions and time. Differential expression of
the reporter gene between samples exposed to the agent and control
samples identifies agents which modulate the expression of the
nucleic acid.
[0097] Additional assay formats may be used to monitor the ability
of the agent to modulate the expression of a gene identified in
Tables 5A-5XX. For instance, as described above, mRNA expression
may be monitored directly by hybridization of probes to the nucleic
acids of the invention. Cell lines are exposed to the agent to be
tested under appropriate conditions and time and total RNA or mRNA
is isolated by standard procedures such those disclosed in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 3d Ed. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001).
[0098] In another assay format, cells or cell lines are first
identified which express the gene products of the invention
physiologically. Cell and/or cell lines so identified would be
expected to comprise the necessary cellular machinery such that the
fidelity of modulation of the transcriptional apparatus is
maintained with regard to exogenous contact of agent with
appropriate surface transduction mechanisms and/or the cytosolic
cascades. Further, such cells or cell lines may be transduced or
transfected with an expression vehicle (e.g., a plasmid or viral
vector) construct comprising an operable non-translated 5'-promoter
containing end of the structural gene encoding the gene products of
Tables 5A-5XX fused to one or more antigenic fragments or other
detectable markers, which are peculiar to the instant gene
products, wherein said fragments are under the transcriptional
control of said promoter and are expressed as polypeptides whose
molecular weight can be distinguished from the naturally occurring
polypeptides or may further comprise an immunologically distinct or
other detectable tag. Such a process is well known in the art (see
Sambrook et al., supra).
[0099] Cells or cell lines transduced or transfected as outlined
above are then contacted with agents under appropriate conditions;
for example, the agent comprises a pharmaceutically acceptable
excipient and is contacted with cells comprised in an aqueous
physiological buffer such as phosphate buffered saline (PBS) at
physiological pH, Eagles balanced salt solution (BSS) at
physiological pH, PBS or BSS comprising serum or conditioned media
comprising PBS or BSS and/or serum incubated at 37.degree. C. Said
conditions may be modulated as deemed necessary by one of skill in
the art. Subsequent to contacting the cells with the agent, said
cells are disrupted and the polypeptides of the lysate are
fractionated such that a polypeptide fraction is pooled and
contacted with an antibody to be further processed by immunological
assay (e.g., ELISA, immunoprecipitation or Western blot). The pool
of proteins isolated from the agent-contacted sample is then
compared with the control samples (no exposure and exposure to a
known toxin) where only the excipient is contacted with the cells
and an increase or decrease in the immunologically generated signal
from the agent-contacted sample compared to the control is used to
distinguish the effectiveness and/or toxic effects of the
agent.
[0100] Another embodiment of the present invention provides methods
for identifying agents that modulate at least one activity of a
protein(s) encoded by the genes in Tables 5A-5XX. Such methods or
assays may utilize any means of monitoring or detecting the desired
activity.
[0101] In one format, the relative amounts of a protein (Tables
5A-5XX) between a cell population that has been exposed to the
agent to be tested compared to an unexposed control cell population
and a cell population exposed to a known toxin may be assayed. In
this format, probes such as specific antibodies are used to monitor
the differential expression of the protein in the different cell
populations. Cell lines or populations are exposed to the agent to
be tested under appropriate conditions and time. Cellular lysates
may be prepared from the exposed cell line or population and a
control, unexposed cell line or population. The cellular lysates
are then analyzed with the probe, such as a specific antibody.
[0102] Agents that are assayed in the above methods can be randomly
selected or rationally selected or designed. As used herein, an
agent is said to be randomly selected when the agent is chosen
randomly without considering the specific sequences involved in the
association of the a protein of the invention alone or with its
associated substrates, binding partners, etc. An example of
randomly selected agents is the use a chemical library or a peptide
combinatorial library, or a growth broth of an organism.
[0103] As used herein, an agent is said to be rationally selected
or designed when the agent is chosen on a nonrandom basis which
takes into account the sequence of the target site and/or its
conformation in connection with the agent's action. Agents can be
rationally selected or rationally designed by utilizing the peptide
sequences that make up these sites. For example, a rationally
selected peptide agent can be a peptide whose amino acid sequence
is identical to or a derivative of any functional consensus
site.
[0104] The agents of the present invention can be, as examples,
peptides, small molecules, vitamin derivatives, as well as
carbohydrates. Dominant negative proteins, DNAs encoding these
proteins, antibodies to these proteins, peptide fragments of these
proteins or mimics of these proteins may be introduced into cells
to affect function. "Mimic" used herein refers to the modification
of a region or several regions of a peptide molecule to provide a
structure chemically different from the parent peptide but
topographically and functionally similar to the parent peptide (see
G. A. Grant in: Molecular Biology and Biotechnology, Meyers, ed.,
pp. 659-664, VCH Publishers, New York, 1995). A skilled artisan can
readily recognize that there is no limit as to the structural
nature of the agents of the present invention.
Nucleic Acid Assay Formats
[0105] The genes identified as being differentially expressed upon
exposure to a known hepatotoxin (Tables 5A-5XX) may be used in a
variety of nucleic acid detection assays to detect or quantititate
the expression level of a gene or multiple genes in a given sample.
The genes described in Tables 5A-5XX may also be used in
combination with one or more additional genes whose differential
expression is associate with toxicity in a cell or tissue. In
preferred embodiments, the genes in Tables 5A-5XX may be combined
with one or more of the genes described in prior and related
applications 60/353,171; 60/363,534; 60/371,135; 60/371,134;
60/370,248; 60/371,150; 60/371,413; 60/373,601; 60/374,139;
60/394,253; 60/378,652; 60/373,602; 60/378,653; 60/378,665;
60/378,370; 60/394,230; 60/407,688; 09/917,800; 10/060,087;
PCT/US03/______, entitled "Molecular Hepatotoxicology Modeling,"
filed Jan. 31, 2003; and PCT/US01/23872, all of which are
incorporated by reference on page 1 of this application.
[0106] Any assay format to detect gene expression may be used. For
example, traditional Northern blotting, dot or slot blot, nuclease
protection, primer directed amplification, RT-PCR, semi- or
quantitative PCR, branched-chain DNA and differential display
methods may be used for detecting gene expression levels. Those
methods are useful for some embodiments of the invention. In cases
where smaller numbers of genes are detected, amplification based
assays may be most efficient. Methods and assays of the invention,
however, may be most efficiently designed with hybridization-based
methods for detecting the expression of a large number of
genes.
[0107] Any hybridization assay format may be used, including
solution-based and solid support-based assay formats. Solid
supports containing oligonucleotide probes for differentially
expressed genes of the invention can be filters, polyvinyl chloride
dishes, particles, beads, microparticles or silicon or glass based
chips, etc. Such chips, wafers and hybridization methods are widely
available, for example, those disclosed by Beattie (WO
95/11755).
[0108] Any solid surface to which oligonucleotides can be bound,
either directly or indirectly, either covalently or non-covalently,
can be used. A preferred solid support is a high density array or
DNA chip. These contain a particular oligonucleotide probe in a
predetermined location on the array. Each predetermined location
may contain more than one molecule of the probe, but each molecule
within the predetermined location has an identical sequence. Such
predetermined locations are termed features. There may be, for
example, from 2, 10, 100, 1000 to 10,000, 100,000 or 400,000 or
more of such features on a single solid support. The solid support,
or the area within which the probes are attached may be on the
order of about a square centimeter. Probes corresponding to the
genes of Tables 5A-5XX or from the related applications described
above may be attached to single or multiple solid support
structures, e.g., the probes may be attached to a single chip or to
multiple chips to comprise a chip set.
[0109] Oligonucleotide probe arrays for expression monitoring can
be made and used according to any techniques known in the art (see
for example, Lockhart et al., Nat Biotechnol 14:1675-1680 (1996);
McGall et al., Proc Nat Acad Sci USA 93:13555-13460 (1996)). Such
probe arrays may contain at least two or more oligonucleotides that
are complementary to or hybridize to two or more of the genes
described in Tables 5A-5XX. For instance, such arrays may contain
oligonucleotides that are complementary or hybridize to at least 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 70, 100 or more the genes
described herein. Preferred arrays contain all or nearly all of the
genes listed in Tables 5A-5XX, or individually, the gene sets of
Tables 5A-5XX. In a preferred embodiment, arrays are constructed
that contain oligonucleotides to detect all or nearly all of the
genes in any one of or all of Tables 5A-5XX on a single solid
support substrate, such as a chip.
[0110] The sequences of the expression marker genes of Tables
5A-5XX are in the public databases. Table 1 provides the GenBank
Accession Number, SEQ ID NO: and GLGC ID No. (Gene Logic reference
no.) for each of the sequences (see www.ncbi.nlm.nih.gov/), while
Table 2 provides identification information for the human
homologues of the genes of Tables 1 and 5A-5XX. Table 3 identifies
the metabolic pathways in which the genes of Tables 1 and 5A-5XX
are believed to function. Table 4 defines the model codes used in
Tables 1, 2, 3 and 5A-5XX. The sequences of the genes in GenBank
are expressly herein incorporated by reference in their entirety as
of the filing date of this application, as are related sequences,
for instance, sequences from the same gene of different lengths,
variant sequences, polymorphic sequences, genomic sequences of the
genes and related sequences from different species, including the
human counterparts, where appropriate. These sequences may be used
in the methods of the invention or may be used to produce the
probes and arrays of the invention. In some embodiments, the genes
in Tables 5A-5XX that correspond to the genes or fragments
previously associated with a toxic response may be excluded from
the Tables.
[0111] As described above, in addition to the sequences of the
GenBank Accession Nos. and GLGC ID Nos. disclosed in the Tables
5A-5XX, sequences such as naturally occurring variant or
polymorphic sequences may be used in the methods and compositions
of the invention. For instance, expression levels of various
allelic or homologous forms of a gene disclosed in the Tables
5A-5XX may be assayed. Any and all nucleotide variations that do
not alter the functional activity of a gene listed in the Tables
5A-5XX, including all naturally occurring allelic variants of the
genes herein disclosed, may be used in the methods and to make the
compositions (e.g., arrays) of the invention.
[0112] Probes based on the sequences of the genes described above
may be prepared by any commonly available method. Oligonucleotide
probes for screening or assaying a tissue or cell sample are
preferably of sufficient length to specifically hybridize only to
appropriate, complementary genes or transcripts. Typically the
oligonucleotide probes will be at least about 10, 12, 14, 16, 18,
20 or 25 nucleotides in length. In some cases, longer probes of at
least 30, 40, or 50 nucleotides will be desirable.
[0113] As used herein, oligonucleotide sequences that are
complementary to one or more of the genes described in Tables
5A-5XX refer to oligonucleotides that are capable of hybridizing
under stringent conditions to at least part of the nucleotide
sequences of said genes. Such hybridizable oligonucleotides will
typically exhibit at least about 75% sequence identity at the
nucleotide level to said genes, preferably about 80% or 85%
sequence identity or more preferably about 90% or 95% or more
sequence identity to said genes.
[0114] "Bind(s) substantially" refers to complementary
hybridization between a probe nucleic acid and a target nucleic
acid and embraces minor mismatches that can be accommodated by
reducing the stringency of the hybridization media to achieve the
desired detection of the target polynucleotide sequence.
[0115] The terms "background" or "background signal intensity"
refer to hybridization signals resulting from non-specific binding,
or other interactions, between the labeled target nucleic acids and
components of the oligonucleotide array (e.g., the oligonucleotide
probes, control probes, the array substrate, etc.). Background
signals may also be produced by intrinsic fluorescence of the array
components themselves. A single background signal can be calculated
for the entire array, or a different background signal may be
calculated for each target nucleic acid. In a preferred embodiment,
background is calculated as the average hybridization signal
intensity for the lowest 5% to 10% of the probes in the array, or,
where a different background signal is calculated for each target
gene, for the lowest 5% to 10% of the probes for each gene. Of
course, one of skill in the art will appreciate that where the
probes to a particular gene hybridize well and thus appear to be
specifically binding to a target sequence, they should not be used
in a background signal calculation. Alternatively, background may
be calculated as the average hybridization signal intensity
produced by hybridization to probes that are not complementary to
any sequence found in the sample (e.g. probes directed to nucleic
acids of the opposite sense or to genes not found in the sample
such as bacterial genes where the sample is mammalian nucleic
acids). Background can also be calculated as the average signal
intensity produced by regions of the array that lack any probes at
all.
[0116] The phrase "hybridizing specifically to" refers to the
binding, duplexing, or hybridizing of a molecule substantially to
or only to a particular nucleotide sequence or sequences under
stringent conditions when that sequence is present in a complex
mixture (e.g., total cellular) DNA or RNA.
[0117] Assays and methods of the invention may utilize available
formats to simultaneously screen at least about 100, preferably
about 1000, more preferably about 10,000 and most preferably about
1,000,000 different nucleic acid hybridizations.
[0118] As used herein a "probe" is defined as a nucleic acid,
capable of binding to a target nucleic acid of complementary
sequence through one or more types of chemical bonds, usually
through complementary base pairing, usually through hydrogen bond
formation. As used herein, a probe may include natural (i.e., A, G,
U, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In
addition, the bases in probes may be joined by a linkage other than
a phosphodiester bond, so long as it does not interfere with
hybridization. Thus, probes may be peptide nucleic acids in which
the constituent bases are joined by peptide bonds rather than
phosphodiester linkages.
[0119] The term "perfect match probe" refers to a probe that has a
sequence that is perfectly complementary to a particular target
sequence. The test probe is typically perfectly complementary to a
portion (subsequence) of the target sequence. The perfect match
(PM) probe can be a "test probe", a "normalization control" probe,
an expression level control probe and the like. A perfect match
control or perfect match probe is, however, distinguished from a
"mismatch control" or "mismatch probe."
[0120] The terms "mismatch control" or "mismatch probe" refer to a
probe whose sequence is deliberately selected not to be perfectly
complementary to a particular target sequence. For each mismatch
(MM) control in a high-density array there typically exists a
corresponding perfect match (PM) probe that is perfectly
complementary to the same particular target sequence. The mismatch
may comprise one or more bases.
[0121] While the mismatch(s) may be located anywhere in the
mismatch probe, terminal mismatches are less desirable as a
terminal mismatch is less likely to prevent hybridization of the
target sequence. In a particularly preferred embodiment, the
mismatch is located at or near the center of the probe such that
the mismatch is most likely to destabilize the duplex with the
target sequence under the test hybridization conditions.
[0122] The term "stringent conditions" refers to conditions under
which a probe will hybridize to its target subsequence, but with
only insubstantial hybridization to other sequences or to other
sequences such that the difference may be identified. Stringent
conditions are sequence-dependent and will be different in
different circumstances. Longer sequences hybridize specifically at
higher temperatures. Generally, stringent conditions are selected
to be about 5.degree. C. lower than the thermal melting point (Tm)
for the specific sequence at a defined ionic strength and pH.
[0123] Typically, stringent conditions will be those in which the
salt concentration is at least about 0.01 to 1.0 M Na.sup.+ ion
concentration (or other salts) at pH 7.0 to 8.3 and the temperature
is at least about 30.degree. C. for short probes (e.g., 10 to 50
nucleotides). Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide.
[0124] The "percentage of sequence identity" or "sequence identity"
is determined by comparing two optimally aligned sequences or
subsequences over a comparison window or span, wherein the portion
of the polynucleotide sequence in the comparison window may
optionally comprise additions or deletions (i.e., gaps) as compared
to the reference sequence (which does not comprise additions or
deletions) for optimal alignment of the two sequences. The
percentage is calculated by determining the number of positions at
which the identical submit (e.g. nucleic acid base or amino acid
residue) occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the window of comparison and multiplying the
result by 100 to yield the percentage of sequence identity.
Percentage sequence identity when calculated using the programs GAP
or BESTFIT (see below) is calculated using default gap weights.
Probe Design
[0125] One of skill in the art will appreciate that an enormous
number of array designs are suitable for the practice of this
invention. The high density array will typically include a number
of test probes that specifically hybridize to the sequences of
interest. Probes may be produced from any region of the genes
identified in the Tables and the attached representative sequence
listing. In instances where the gene reference in the Tables is an
EST, probes may be designed from that sequence or from other
regions of the corresponding full-length transcript that may be
available in any of the sequence databases, such as those herein
described. See WO 99/32660 for methods of producing probes for a
given gene or genes. In addition, any available software may be
used to produce specific probe sequences, including, for instance,
software available from Molecular Biology Insights, Olympus Optical
Co. and Biosoft International. In a preferred embodiment, the array
will also include one or more control probes.
[0126] High density array chips of the invention include "test
probes." Test probes may be oligonucleotides that range from about
5 to about 500, or about 7 to about 50 nucleotides, more preferably
from about 10 to about 40 nucleotides and most preferably from
about 15 to about 35 nucleotides in length. In other particularly
preferred embodiments, the probes are 20 or 25 nucleotides in
length. In another preferred embodiment, test probes are double or
single strand DNA sequences. DNA sequences are isolated or cloned
from natural sources or amplified from natural sources using native
nucleic acid as templates. These probes have sequences
complementary to particular subsequences of the genes whose
expression they are designed to detect. Thus, the test probes are
capable of specifically hybridizing to the target nucleic acid they
are to detect.
[0127] In addition to test probes that bind the target nucleic
acid(s) of interest, the high density array can contain a number of
control probes. The control probes may fall into three categories
referred to herein as 1) normalization controls; 2) expression
level controls; and 3) mismatch controls.
[0128] Normalization controls are oligonucleotide or other nucleic
acid probes that are complementary to labeled reference
oligonucleotides or other nucleic acid sequences that are added to
the nucleic acid sample to be screened. The signals obtained from
the normalization controls after hybridization provide a control
for variations in hybridization conditions, label intensity,
"reading" efficiency and other factors that may cause the signal of
a perfect hybridization to vary between arrays. In a preferred
embodiment, signals (e.g., fluorescence intensity) read from all
other probes in the array are divided by the signal (e.g.,
fluorescence intensity) from the control probes thereby normalizing
the measurements.
[0129] Virtually any probe may serve as a normalization control.
However, it is recognized that hybridization efficiency varies with
base composition and probe length. Preferred normalization probes
are selected to reflect the average length of the other probes
present in the array, however, they can be selected to cover a
range of lengths. The normalization control(s) can also be selected
to reflect the (average) base composition of the other probes in
the array, however in a preferred embodiment, only one or a few
probes are used and they are selected such that they hybridize well
(i.e., no secondary structure) and do not match any target-specific
probes.
[0130] Expression level controls are probes that hybridize
specifically with constitutively expressed genes in the biological
sample. Virtually any constitutively expressed gene provides a
suitable target for expression level controls. Typically expression
level control probes have sequences complementary to subsequences
of constitutively expressed "housekeeping genes" including, but not
limited to the .beta.-actin gene, the glyceraldehyde-3-phosphate
dehydrogenase (GADPH) gene, the transferrin receptor gene and the
like.
[0131] Mismatch controls may also be provided for the probes to the
target genes, for expression level controls or for normalization
controls. Mismatch controls are oligonucleotide probes or other
nucleic acid probes identical to their corresponding test or
control probes except for the presence of one or more mismatched
bases. A mismatched base is a base selected so that it is not
complementary to the corresponding base in the target sequence to
which the probe would otherwise specifically hybridize. One or more
mismatches are selected such that under appropriate hybridization
conditions (e.g., stringent conditions) the test or control probe
would be expected to hybridize with its target sequence, but the
mismatch probe would not hybridize (or would hybridize to a
significantly lesser extent) Preferred mismatch probes contain a
central mismatch. Thus, for example, where a probe is a 20 mer, a
corresponding mismatch probe will have the identical sequence
except for a single base mismatch (e.g., substituting a G, a C or a
T for an A) at any of positions 6 through 14 (the central
mismatch).
[0132] Mismatch probes thus provide a control for non-specific
binding or cross hybridization to a nucleic acid in the sample
other than the target to which the probe is directed. For example,
if the target is present the perfect match probes should be
consistently brighter than the mismatch probes. In addition, if all
central mismatches are present, the mismatch probes can be used to
detect a mutation, for instance, a mutation of a gene in the
accompanying Tables 5A-5XX. The difference in intensity between the
perfect match and the mismatch probe provides a good measure of the
concentration of the hybridized material.
Nucleic Acid Samples
[0133] Cell or tissue samples may be exposed to the test agent in
vitro or in vivo. When cultured cells or tissues are used,
appropriate mammalian liver extracts may also be added with the
test agent to evaluate agents that may require biotransformation to
exhibit toxicity. In a preferred format, primary isolates of animal
or human hepatocytes which already express the appropriate
complement of drug-metabolizing enzymes may be exposed to the test
agent without the addition of mammalian liver extracts.
[0134] The genes which are assayed according to the present
invention are typically in the form of mRNA or reverse transcribed
mRNA. The genes may be cloned or not. The genes may be amplified or
not. The cloning and/or amplification do not appear to bias the
representation of genes within a population. In some assays, it may
be preferable, however, to use polyA+ RNA as a source, as it can be
used with less processing steps.
[0135] As is apparent to one of ordinary skill in the art, nucleic
acid samples used in the methods and assays of the invention may be
prepared by any available method or process. Methods of isolating
total mRNA are well known to those of skill in the art. For
example, methods of isolation and purification of nucleic acids are
described in detail in Chapter 3 of Laboratory Techniques in
Biochemistry and Molecular Biology Vol. 24, Hybridization With
Nucleic Acid Probes: Theory and Nucleic Acid Probes, P. Tijssen,
Ed., Elsevier Press, New York, 1993. Such samples include RNA
samples, but also include cDNA synthesized from a mRNA sample
isolated from a cell or tissue of interest. Such samples also
include DNA amplified from the cDNA, and RNA transcribed from the
amplified DNA. One of skill in the art would appreciate that it is
desirable to inhibit or destroy RNase present in homogenates before
homogenates are used.
[0136] Biological samples may be of any biological tissue or fluid
or cells from any organism as well as cells raised in vitro, such
as cell lines and tissue culture cells. Frequently the sample will
be a tissue or cell sample that has been exposed to a compound,
agent, drug, pharmaceutical composition, potential environmental
pollutant or other composition. In some formats, the sample will be
a "clinical sample" which is a sample derived from a patient.
Typical clinical samples include, but are not limited to, sputum,
blood, blood-cells (e.g., white cells), tissue or fine needle
biopsy samples, urine, peritoneal fluid, and pleural fluid, or
cells therefrom.
[0137] Biological samples may also include sections of tissues,
such as frozen sections or formalin fixed sections taken for
histological purposes.
Forming High Density Arrays
[0138] Methods of forming high density arrays of oligonucleotides
with a minimal number of synthetic steps are known. The
oligonucleotide analogue array can be synthesized on a single or on
multiple solid substrates by a variety of methods, including, but
not limited to, light-directed chemical coupling, and mechanically
directed coupling (see Pirrung, U.S. Pat. No. 5,143,854).
[0139] In brief, the light-directed combinatorial synthesis of
oligonucleotide arrays on a glass surface proceeds using automated
phosphoramidite chemistry and chip masking techniques. In one
specific implementation, a glass surface is derivatized with a
silane reagent containing a functional group, e.g., a hydroxyl or
amine group blocked by a photolabile protecting group. Photolysis
through a photolithogaphic mask is used selectively to expose
functional groups which are then ready to react with incoming 5'
photoprotected nucleoside phosphoramidites. The phosphoramidites
react only with those sites which are illuminated (and thus exposed
by removal of the photolabile blocking group). Thus, the
phosphoramidites only add to those areas selectively exposed from
the preceding step. These steps are repeated until the desired
array of sequences have been synthesized on the solid surface.
Combinatorial synthesis of different oligonucleotide analogues at
different locations on the array is determined by the pattern of
illumination during synthesis and the order of addition of coupling
reagents.
[0140] In addition to the foregoing, additional methods which can
be used to generate an array of oligonucleotides on a single
substrate are described in PCT Publication Nos. WO 93/09668 and WO
01/23614. High density nucleic acid arrays can also be fabricated
by depositing pre-made or natural nucleic acids in predetermined
positions. Synthesized or natural nucleic acids are deposited on
specific locations of a substrate by light directed targeting and
oligonucleotide directed targeting. Another embodiment uses a
dispenser that moves from region to region to deposit nucleic acids
in specific spots.
Hybridization
[0141] Nucleic acid hybridization simply involves contacting a
probe and target nucleic acid under conditions where the probe and
its complementary target can form stable hybrid duplexes through
complementary base pairing. See WO 99/32660. The nucleic acids that
do not form hybrid duplexes are then washed away leaving the
hybridized nucleic acids to be detected, typically through
detection of an attached detectable label. It is generally
recognized that nucleic acids are denatured by increasing the
temperature or decreasing the salt concentration of the buffer
containing the nucleic acids. Under low stringency conditions
(e.g., low temperature and/or high salt) hybrid duplexes (e.g.,
DNA:DNA, RNA:RNA, or RNA:DNA) will form even where the annealed
sequences are not perfectly complementary. Thus, specificity of
hybridization is reduced at lower stringency. Conversely, at higher
stringency (e.g., higher temperature or lower salt) successful
hybridization tolerates fewer mismatches. One of skill in the art
will appreciate that hybridization conditions may be selected to
provide any degree of stringency.
[0142] In a preferred embodiment, hybridization is performed at low
stringency, in this case in 6.times.SSPET at 37.degree. C. (0.005%
Triton X-100), to ensure hybridization and then subsequent washes
are performed at higher stringency (e.g., 1.times.SSPET at
37.degree. C.) to eliminate mismatched hybrid duplexes. Successive
washes may be performed at increasingly higher stringency (e.g.,
down to as low as 0.25.times.SSPET at 37.degree. C. to 50.degree.
C.) until a desired level of hybridization specificity is obtained.
Stringency can also be increased by addition of agents such as
formamide. Hybridization specificity may be evaluated by comparison
of hybridization to the test probes with hybridization to the
various controls that can be present (e.g., expression level
control, normalization control, mismatch controls, etc.).
[0143] In general, there is a tradeoff between hybridization
specificity (stringency) and signal intensity. Thus, in a preferred
embodiment, the wash is performed at the highest stringency that
produces consistent results and that provides a signal intensity
greater than approximately 10% of the background intensity. Thus,
in a preferred embodiment, the hybridized array may be washed at
successively higher stringency solutions and read between each
wash. Analysis of the data sets thus produced will reveal a wash
stringency above which the hybridization pattern is not appreciably
altered and which provides adequate signal for the particular
oligonucleotide probes of interest.
Signal Detection
[0144] The hybridized nucleic acids are typically detected by
detecting one or more labels attached to the sample nucleic acids.
The labels may be incorporated by any of a number of means well
known to those of skill in the art. See WO 99/32660.
Databases
[0145] The present invention includes relational databases
containing sequence information, for instance, for the genes of
Tables 5A-5XX, as well as gene expression information from tissue
or cells exposed to various standard toxins, such as those herein
described (see Tables 5A-5XX). Databases may also contain
information associated with a given sequence or tissue sample such
as descriptive information about the gene associated with the
sequence information (see Tables 1, 2 and 3), or descriptive
information concerning the clinical status of the tissue sample, or
the animal from which the sample was derived. The database may be
designed to include different parts, for instance a sequence
database and a gene expression database. Methods for the
configuration and construction of such databases and
computer-readable media to which such databases are saved are
widely available, for instance, see U.S. Pat. No. 5,953,727, which
is herein incorporated by reference in its entirety.
[0146] The databases of the invention may be linked to an outside
or external database such as GenBank
(www.ncbi.nlm.nih.gov/entrez.index.html); KEGG
(www.genome.ad.jp/kegg); SPAD
(www.grt.kyushu-u.ac.jp/spad/index.html); HUGO
(www.gene.ucl.ac.uk/hugo); Swiss-Prot (www.expasy.ch.sprot);
Prosite (www.expasy.ch/tools/scnpsit1.html); OMIM
(www.ncbi.nlm.nih.gov/omim); LocusLink
(www.ncbi.nlm.nih.gov/LocusLink/); RefSeq
(www.ncbi.nlm.nih.gov/LocusLink/refseq.html) and GDB (www.gdb.org).
In a preferred embodiment, as described in Tables 1-3, the external
database is GenBank and the associated databases maintained by the
National Center for Biotechnology Information (NCBI)
(www.ncbi.nlm.nih.gov).
[0147] Any appropriate computer platform, user interface, etc. may
be used to perform the necessary comparisons between sequence
information, gene expression information and any other information
in the database or information provided as an input. For example, a
large number of computer workstations are available from a variety
of manufacturers, such has those available from Silicon Graphics.
Client/server environments, database servers and networks are also
widely available and appropriate platforms for the databases of the
invention.
[0148] The databases of the invention may be used to produce, among
other things, electronic Northerns that allow the user to determine
the cell type or tissue in which a given gene is expressed and to
allow determination of the abundance or expression level of a given
gene in a particular tissue or cell.
[0149] The databases of the invention may also be used to present
information identifying the expression level in a tissue or cell of
a set of genes comprising one or more of the genes in Tables
5A-5XX, comprising the step of comparing the expression level of at
least one gene in Tables 5A-5XX in a cell or tissue exposed to a
test agent to the level of expression of the gene in the database.
Such methods may be used to predict the toxic potential of a given
compound by comparing the level of expression of a gene or genes in
Tables 5A-5XX from a tissue or cell sample exposed to the test
agent to the expression levels found in a control tissue or cell
samples exposed to a standard toxin or hepatotoxin such as those
herein described. Such methods may also be used in the drug or
agent screening assays as described herein.
Kits
[0150] The invention further includes kits combining, in different
combinations, high-density oligonucleotide arrays, reagents for use
with the arrays, protein reagents encoded by the genes of the
Tables, signal detection and array-processing instruments, gene
expression databases and analysis and database management software
described above. The kits may be used, for example, to predict or
model the toxic response of a test compound, to monitor the
progression of hepatic disease states, to identify genes that show
promise as new drug targets and to screen known and newly designed
drugs as discussed above.
[0151] The databases packaged with the kits are a compilation of
expression patterns from human or laboratory animal genes and gene
fragments (corresponding to the genes of Tables 5A-5XX). In
particular, the database software and packaged information that may
contain the databases saved to a computer-readable medium include
the expression results of Tables 5A-5XX that can be used to predict
toxicity of a test agent by comparing the expression levels of the
genes of Tables 5A-5XX induced by the test agent to the expression
levels presented in Tables 5A-5XX. In another format, database and
software information may be provided in a remote electronic format,
such as a website, the address of which may be packaged in the
kit.
[0152] The kits may used in the pharmaceutical industry, where the
need for early drug testing is strong due to the high costs
associated with drug development, but where bioinformatics, in
particular gene expression informatics, is still lacking. These
kits will reduce the costs, time and risks associated with
traditional new drug screening using cell cultures and laboratory
animals. The results of large-scale drug screening of pre-grouped
patient populations, pharmacogenomics testing, can also be applied
to select drugs with greater efficacy and fewer side-effects. The
kits may also be used by smaller biotechnology companies and
research institutes who do not have the facilities for performing
such large-scale testing themselves.
[0153] Databases and software designed for use with use with
microarrays is discussed in Balaban et al., U.S. Pat. Nos.
6,229,911, a computer-implemented method for managing information,
stored as indexed tables, collected from small or large numbers of
microarrays, and 6,185,561, a computer-based method with data
mining capability for collecting gene expression level data, adding
additional attributes and reformatting the data to produce answers
to various queries. Chee et al., U.S. Pat. No. 5,974,164, discloses
a software-based method for identifying mutations in a nucleic acid
sequence based on differences in probe fluorescence intensities
between wild type and mutant sequences that hybridize to reference
sequences.
[0154] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples therefore, specifically point out the
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the
disclosure.
EXAMPLES
Example 1
Identification of Toxicity Markers in Rat Hepatocytes
[0155] To evaluate their toxicity, the hepatotoxins
alpha-naphthylisothiocyante (ANIT), acetaminophen (APAP), AY-25329,
carbon tetrachloride, clofibrate, diclofenac,
17.alpha.-ethinylestradiol, hydrazine, indomethacin,
lipopolysaccharide, lovastatin, methotrexate, tacrine, valproate
and control compositions were administered to cultures of primary
rat hepatocytes from male Sprague-Dawley rats at various time
points using administration diluents, protocols and dosing regimes
as previously described in the art and in the prior applications
discussed above, as well as in Table 6. Laboratory protocols for
the administration of the hepatotoxins amiodarone, carbamazepine,
chlorpromazine, CI-1000, CPA, diflunisal, DMN, gemfibrozil,
imipramine, phenobarbital, tamoxifen, tetracycline and Wy-14643
also appear in Table 6. Identification of toxicity markers was
performed by microarray analysis and by the AlamarBlue.RTM. assay,
a classical measure of cytotoxicity. The AlamarBlue.RTM. assay was
performed in triplicate.
[0156] The source of the primary rat hepatocytes was Sprague Dawley
Outbred CD.RTM. Rats (CRL:CD.RTM.[SD] IGS BR, Charles River
Laboratories). Hepatocyte cultures were obtained in 24-well
matrigel coated plates for the AlamarBlue.RTM. assay (175,000
cells/cm.sup.2) or in T-75 cm.sup.2 matrigel coated flasks for RNA
isolation for microarray analysis (187,000 cells/cm.sup.2). Primary
rat hepatocytes were received the day after the cells were removed
from the animals. After arrival, the cells, the cells were
incubated overnight (.about.15 hrs) before the toxin was added to
the cultures. The vehicle used in the toxicity experiments was HIM
culture medium (Hepatocyte Incubation Medium, In Vitro Technologies
Cat. No. Z90009) containing 0.2% DMSO (Sigma Cat. No. D-5879).
Toxin or vehicle was administered to hepatocyte cultures as
follows. For each treatment, i.e., vehicle alone, vehicle+toxin at
low dose, or vehicle+toxin at high dose, cells were harvested after
3, 6 and 24-hour incubations with the toxin solution or with the
vehicle.
[0157] The AlamarBlue.RTM. assay was performed as follows, using
only the 24-hour time point samples. [0158] 1. Primary rat
hepatocyte cultures were prepared as described above in a
matrigel-coated plates at 175,000 cells/cm.sup.2. [0159] 2. The
culture medium (HIM) was removed from each well and replaced with
500 .mu.l of fresh HIM following arrival of the cells, and the
cells were incubated overnight (approximately 15 hrs) at 37.degree.
C., 5% CO.sub.2. [0160] 3. The next day, the HIM was removed and
500 .mu.l of the medium containing either vehicle or a dose of
toxin was added. [0161] 4. Lysis solution was used as a negative
control. 450 .mu.l medium+50 .mu.l 19% Triton X100 were added to
each of 3 wells containing cells, for a final Triton concentration
of 1%. [0162] 5. The cells in all wells were incubated for 24 hours
at 37.degree. C., 5% CO.sub.2. [0163] 6. HIM medium was removed,
and a solution containing 500 .mu.l of fresh HIM medium+50 .mu.l
AlamarBlue.RTM. (BioSource International, Inc., Cat. No. DAL1100)
was added to each well. [0164] 7. The cells were incubated at
37.degree. C., 5% CO.sub.2 for 2 hours. [0165] 8. 100 .mu.l medium
was removed from each well of the 24-well plate and added to a well
of a 96-well plate. The fluorescence was measured using 544 nm as
the excitation and 590 um as the emission on a Molecular Devices,
SpectraMax Gemini, Softmax pro 2.6.1. Alternatively, two absorbance
readings can be measured for the oxidized (600 nm) and the reduced
(570 nm) form of AlamarBlue.RTM.. After obtaining absorbance
readings, results were calculated according to the manufacturer's
protocol provided in the product description. [0166] 9. The data
were evaluated to determine whether or not the toxin reduced cell
viability. If so, the dose of the toxin that reduced cell viability
by .about.10-20% was determined. Collection of RNA from Rat
Hepatocytes
[0167] More than 10.sup.7 cells are typically prepared for each
sample. RNA was collected at 3, 6 and 24 hours following addition
of the toxin according to the following procedure.
[0168] The medium from the flasks was discarded, and the cells were
washed once with 20 ml of warm (37.degree. C.) RPMI-1640+10 mM
HEPES medium (Life Technologies, Cat. No. 22400-089). 12 ml of
Trizol (Life Technologies, Cat. No. 15596-018) was placed
immediately into each T-75 flask. Each flask contained .about.10-20
million cells. The contents of each flask were mixed vigorously for
one minute with a vortex mixer and then aspirated up and down 5
times with a pipette. The contents of each flask (.about.12 ml
each) was collected into a 50 ml conical polypropylene tissue
culture tube (Falcon), snap frozen in liquid nitrogen and stored at
.ltoreq.-86.degree. C.
[0169] Microarray sample preparation was conducted with minor
modifications, following the protocols set forth in the Affymetrix
GeneChip.RTM. Expression Analysis Manual. Frozen cells were ground
to a powder using a Spex Certiprep 6800 Freezer Mill. Total RNA was
extracted with Trizol (GibcoBRL) utilizing the manufacturer's
protocol. The total RNA yield for each sample was 200-500 .mu.g per
300 mg cells. mRNA was isolated using the Oligotex mRNA Midi kit
(Qiagen) followed by ethanol precipitation. Double stranded cDNA
was generated from mRNA using the SuperScript Choice system
(GibcoBRL). First strand cDNA synthesis was primed with a T7-(dT24)
oligonucleotide. The cDNA was phenol-chloroform extracted and
ethanol precipitated to a final concentration of 1 .mu.g/ml. From 2
.mu.g of cDNA, cRNA was synthesized using Ambion's T7 MegaScript in
vitro Transcription Kit.
[0170] To biotin label the cRNA, nucleotides Bio-11-CTP and
Bio-16-UTP (Enzo Diagnostics) were added to the reaction. Following
a 37.degree. C. incubation for six hours, impurities were removed
from the labeled cRNA following the RNeasy Mini kit protocol
(Qiagen). cRNA was fragmented (fragmentation buffer consisting of
200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc) for
thirty-five minutes at 94.degree. C. Following the Affymetrix
protocol, 55 .mu.g of fragmented cRNA was hybridized on the
Affymetrix rat array set for twenty-four hours at 60 rpm in a
45.degree. C. hybridization oven. The chips were washed and stained
with Streptavidin Phycoerythrin (SAPE) (Molecular Probes) in
Affymetrix fluidics stations. To amplify staining, SAPE solution
was added twice with an anti-streptavidin biotinylated antibody
(Vector Laboratories) staining step in between. Hybridization to
the probe arrays was detected by fluorometric scanning (Hewlett
Packard Gene Array Scanner). Data was analyzed using Affymetrix
GeneChip.RTM. version 3.0 and Expression Data Mining Tool (EDMT)
software (version 1.0), S-Plus, and the GeneExpress.RTM. software
system.
[0171] Differential expression of genes between the toxin-exposed
and control samples corresponding to patterns indicative of
toxicity was determined using the following criteria.
[0172] Table 1 discloses those genes that are differentially
expressed upon exposure to the named toxins with their
corresponding SEQ ID NOS: GenBank Accession or RefSeq ID Nos., GLGC
ID Nos. (internal Gene Logic identification nos.), gene names and
Unigene Sequence Cluster titles. The metabolic pathways in which
the genes of Table 1 function are indicated in Table 3, and the
corresponding human homologues are given in Table 2. The model
codes, identified in Table 4, represent the various toxicity or
liver pathology states associated with differential expression of
each gene, as well as the individual toxin types associated with
differential expression of each gene.
[0173] Tables 5A-5XX disclose the summary statistics for each of
the comparisons performed. Each of these tables contains a set of
predictive genes and creates a model for predicting the
hepatoxicity of an unknown, i.e., untested compound. Each gene is
identified by its Gene Logic identification number and can be
cross-referenced to a gene name and representative SEQ ID NO. in
Table 1. For each comparison of gene expression levels between
samples in the toxicity group ("Tox" samples, i.e., samples
affected by exposure to a specific toxin) and samples in the
non-toxicity group ("Non-tox" samples, i.e., samples not affected
by exposure to that same specific toxin), the group mean for Tox
samples is the mean signal intensity, as normalized for the various
chip parameters that are being assayed. The Non-tox mean represents
the mean signal intensity, as normalized for the various chip
parameters that are being assayed, in samples other than those
treated with the high dose of the specific toxin. These samples
were treated with a low dose of the specific toxin, or with vehicle
alone, or with a different toxin. Tox samples were obtained from
treated cells processed at the timepoint(s) indicated in the
tables, while Non-tox samples were obtained from control cells
processed at all time points in the experiments. For individual
genes, an increase in the Tox group mean compared to the Non-tox
group mean indicates up-regulation upon exposure to a toxin.
Conversely, a decrease in the Tox group mean compared to the
Non-tox group mean indicates down-regulation.
[0174] The mean values are derived from Average Difference
(AveDiff) values for a particular gene, averaged across the
corresponding samples. Each individual Average Difference value is
calculated by integrating the intensity information from multiple
probe pairs that are tiled for a particular fragment. The
normalization multiplies each expression intensity for a given
experiment (chip) by a global scaling factor. The intent of this
normalization is to make comparisons of individual genes between
chips possible. The scaling factor is calculated as follows:
1. From all the unnormalized expression values in the experiment,
delete the largest 2% and smallest 2% of the values. That is, if
the experiment yields 10,000 expression values, order the values
and delete the smallest 200 and the largest 200. 2. Compute the
trimmed mean, which is equal to the mean of the remaining values.
3. Compute the scale factor SF=100/(trimmed mean)
[0175] The value of 100 used here is the standard target value
used. Some AveDiff values may be negative due to the general noise
involved in nucleic acid hybridization experiments. Although many
conclusions can be made corresponding to a negative value on the
GeneChip platform, it is difficult to assess the meaning behind the
negative value for individual fragments. Our observations show
that, although negative values are observed at times within the
predictive gene set, these values reflect a real biological
phenomenon that is highly reproducible across all the samples from
which the measurement was taken. For this reason, those genes that
exhibit a negative value are included in the predictive set. It
should be noted that other platforms of gene expression measurement
may be able to resolve the negative numbers for the corresponding
genes. The predictive ability of each of those genes should extend
across platforms, however. Each mean value is accompanied by the
standard deviation for the mean.
[0176] The linear discriminant analysis score (discriminant score),
as disclosed in the tables, measures the ability of each gene to
predict whether or not a sample is toxic. The discriminant score is
calculated by the following steps:
Calculation of a Discriminant Score
[0177] Let X.sub.i represent the AveDiff values for a given gene
across the Group 1 samples, i=1 . . . n.
[0178] Let Y.sub.i represent the AveDiff values for a given gene
across the Group 2 samples, i=1 . . . t.
[0179] The calculations proceed as follows:
[0180] Calculate mean and standard deviation for X.sub.i's and
Y.sub.i's, and denote these by m.sub.X, m.sub.Y, s.sub.X,
s.sub.Y.
[0181] For all X.sub.i's and Y.sub.i's, evaluate the function
f(z)=((1/sy)*exp(-0.5*((z-m.sub.Y)/s.sub.Y).sup.2))/(((1/s.sub.Y)*exp(-0.-
5*((z-m.sub.Y)/s.sub.Y).sup.2))+((1/s.sub.X)*exp(-0.5*((z-m.sub.X)/s.sub.X-
).sup.2))).
[0182] The number of correct predictions, say P, is then the number
of Y.sub.i's such that f(Y.sub.i)>0.5 plus the number of
X.sub.i's such that f(X.sub.i)<0.5.
[0183] The discriminant score is then P/(n+t).
[0184] Linear discriminant analysis uses both the individual
measurements of each gene and the calculated measurements of all
combinations of genes to classify samples. For each gene, a weight
is derived from the mean and standard deviation of the Tox and
Non-tox sample groups. Every gene is multiplied by a weight and the
sum of these values results in a collective discriminate score.
This discriminant score is then compared against collective
centroids of the Tox and Non-tox groups. These centroids are the
average of all tox and nontox samples respectively. Therefore, each
gene contributes to the overall prediction. This contribution is
dependent on weights that are large positive or negative numbers if
the relative distances between the Tox and Non-tox samples for that
gene are large and small numbers if the relative distances are
small. The discriminant score for each unknown sample and centroid
values can be used to calculate a probability between zero and one
as to the group in which the unknown sample belongs.
Example 2
General Toxicity Modeling
[0185] Samples were selected for grouping into Tox and Non-tox
groups by examining each study individually with Principal
Components Analysis (PCA) to determine which treatments had an
observable response. Only sample groups where confidence of the
tox-responding or non-tox-responding status (expression level
affected by exposure to a specific toxin or expression level not
affected by exposure to a specific toxin, respectively) was
established were included in building a general toxicity prediction
model.
[0186] Linear discriminant models were generated to describe Tox
and Non-tox samples. The top discriminant genes and/or EST's were
used to determine toxicity by calculating each gene's contribution
with homo and heteroscedastic treatment of variance and inclusion
or exclusion of mutual information between genes. Prediction of
samples within the database exceeded 80% true positives with a
false positive rate of less than 5%. It was determined that
combinations of genes and/or EST's generally provided a better
prediction than individual genes and that the more genes and/or EST
used, the better the prediction. Although the preferred embodiment
includes fifty or more genes, many pairings or larger combinations
of genes and/or EST can work better than individual genes. All
combinations of two or more genes from the selected list could be
used to predict toxicity. These combinations could be selected by
pairing in an agglomerate, divisive, or random approach. Further,
as yet undetermined genes and/or EST's could be combined with
individual or a set of genes and/or EST's described here to
increase predictive ability. However, the genes and/or EST's
described here would contribute most of the predictive ability to
any such undetermined combinations.
[0187] Other variations on the above method can provide adequate
predictive ability. These include selective inclusion of components
via agglomerate, divisive, or random approaches or extraction of
loading and combining them in agglomerate, divisive, or random
approaches. Also the use of composite variables in logistic
regression to determine classification of samples can also be
accomplished with linear discriminate analysis, neural or Bayesian
networks, or other forms of regression and classification based on
categorical or continual dependent and independent variables.
Example 3
Modeling Methods
[0188] The above modeling methods provide broad approaches of
combining the expression of genes to predict sample toxicity. One
could also provide no weight in a simple voting method or determine
weights in a supervised or unsupervised method using agglomerate,
divisive, or random approaches. All or selected combinations of
genes may be combined in ordered, agglomerate, or divisive,
supervised or unsupervised clustering algorithms with unknown
samples for classification. Any form of correlation matrix may also
be used to classify unknown samples. The spread of the group
distribution and discriminate score alone provide enough
information to enable a skilled person to generate all of the above
types of models with accuracy that can exceed the discriminate
ability of individual genes. Some examples of methods that could be
used individually or in combination after transformation of data
types include but are not limited to: Discriminant Analysis,
Multiple Discriminant Analysis, logistic regression, multiple
regression analysis, linear regression analysis, conjoint analysis,
canonical correlation, hierarchical cluster analysis, k-means
cluster analysis, self-organizing maps, multidimensional scaling,
structural equation modeling, support vector machine determined
boundaries, factor analysis, neural networks, bayesian
classifications, and resampling methods.
Example 4
Grouping of Individual compound and Pathology Classes
[0189] Samples were grouped into individual pathology classes based
on known toxicological responses and observed clinical chemical and
pathology measurements or into observable toxicity produced by a
compound (Tables 5A-5XX). The top 10, 25, 50, 100 genes based on
individual discriminate scores were used in a model to ensure that
a combination of genes provided a better prediction than individual
genes. As described above, all combinations of two or more genes
from this list could potentially provide better prediction than
individual genes when selected in any order or by ordered,
agglomerate, divisive, or random approaches. In addition, combining
these genes with other genes could provide better predictive
ability, but most of this predictive ability would come from the
genes listed herein.
[0190] A sample may be considered a Tox sample if it scores
positive in any pathological or individual compound class
represented here, or in any modeling method mentioned under general
toxicology models, based on a combination of the sample's time
point and dosage group in a study using an individual compound
(with known or potentially toxic properties) by comparisons
obtainable from the data. The pathological groupings and early and
late phase models are preferred examples of all obtainable
combinations of sample time and dose points. Most logical groupings
with one or more genes and one or more sample dose and time points
should produce better predictions of general toxicity, pathological
specific toxicity, or similarity to a known toxin than individual
genes.
[0191] Although the present invention has been described in detail
with reference to examples above, it is understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims. All cited patents, patent applications and
publications referred to in this application are herein
incorporated by reference in their entirety.
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TABLE-US-00055 TABLE 6 Laboratory protocols for administration of
toxins to hepatocyte cultures and Results of AlamarBlue .RTM. cell
viability assays 3 hours 6 hours 24 hours Toxin low dose high dose
low dose high dose low dose high dose Amiodarone, 6 uM 60 uM 6 uM
60 uM 6 uM 60 uM ICN cat. no. 15353583 % viability by ~100 ~80-85
AlamarBlue test Carbamazepine, 0.01 mM 1 mM 0.01 mM 1 mM 0.01 mM 1
mM Sigma cat. no. C-8981 % viability by ~100 ~90 AlamarBlue test
Chlorpromazine, 3 uM 30 uM 3 uM 30 uM 3 uM 30 uM Sigma cat. no.
C-8138 % viability by ~100 ~75 AlamarBlue test CI-1000 25 uM 250 uM
25 uM 250 uM 25 uM 250 uM % viability by ~90 ~75 AlamarBlue test
Cyproterone acetate 40 uM 400 uM 40 uM 400 uM 40 uM 400 uM Sigma
cat. no. C-3412 % viability by ~100 ~65-70 AlamarBlue test
Diflunisal, 30 uM 300 uM 30 uM 300 uM 30 uM 300 uM Sigma cat. no.
D-3281 % viability by ~100 ~85-90 AlamarBlue test DMN, 1 mM 100 mM
1 mM 100 mM 1 mM 100 mM Sigma cat. no. N-7756 % viability by ~100
~80-85 AlamarBlue test Gemfibrozil, 0.3 mM 3 mM 0.3 mM 3 mM 0.3 mM
3 mM Sigma cat. no. G-9518 % viability by ~100 ~50 AlamarBlue lest
Imipramine, 5 uM 50 uM 5 uM 50 uM 5 uM 50 uM Sigma cat. no. I-7379
% viability by ~100 ~85-90 AlamarBlue test Phenobarbital, 0.8 mM 8
mM 0.8 mM 8 mM 0.8 mM 8 mM Sigma cat. no. P-5178 % viability by
~100 >95 AlamarBlue test Tamoxifen, 4 uM 40 uM 4 uM 40 uM 4 uM
40 uM Sigma cat. no. T-9262 % viability by ~100 ~45 AlamarBlue test
Tetracycline, 0.1 mM 1 mM 0.1 mM 1 mM 0.1 mM 1 mM Sigma cat. no.
T-4062 % viability by ~100 ~85-90 AlamarBlue test Wy-14643 10 uM
100 uM 10 uM 100 uM 10 uM 100 uM Cayman Chem cat. no. 70730 %
viability by ~100 ~90 AlamarBlue test ANIT, 25 uM 250 uM 25 uM 250
uM 25 uM 250 uM Sigma cat. no. N-9883 % viability by ~100 ~60
AlamarBlue test Acetaminophen, 1 mM 10 mM 1 mM 10 mM 1 mM 10 mM
Sigma cat. no. A-7085 % viability by ~100 ~90 AlamarBlue test
AY-25329 5 uM 50 uM 5 uM 50 uM 5 uM 50 uM % viability by ~100 ~90
AlamarBlue test CCl.sub.4, Aldrich 0.1 mM 10 mM 0.1 mM 10 mM 0.1 mM
10 mM cat. no. 31996-1 % viability by ~100 ~80-85 AlamarBlue test
Clofibrate, 0.5 mM 5 mM 0.5 mM 5 mM 0.5 mM 5 mM Sigma cat. no.
C-6643 % viability by ~100 ~80-85 AlamarBlue test Diclofenac, 55 uM
550 uM 55 uM 550 uM 55 uM 550 uM Sigma cat. no. D-6899 % viability
by ~100 ~70 AlamarBlue test 17.alpha.-ethinylestradiol, 10 uM 100
uM 10 uM 100 uM 10 uM 10 uM Sigma cat. no. E-4876 % viability by
~100 ~80 AlamarBlue test Hydrazine, 0.1 mM 1 mM 0.1 mM 1 mM 0.1 mM
1 mM Sigma cat. no. H-0883 % viability by ~90-95 ~80-85 AlamarBlue
test Indomethacin, 0.1 mM 1 mM 0.1 mM 1 mM 0.1 mM 1 mM Sigma cat.
no. 1-8280 % viability by ~100 ~85-90 AlamarBlue test
Lipopolysaccharide, 10 ug/ml 100 ug/ml 10 ug/ml 100 ug/ml 10 ug/ml
100 ug/ml Sigma cat. no. L-8274 % viability by ~100 ~100 AlamarBlue
test Lovastatin, 0.1 mM 1 mM 0.1 mM 1 mM 0.1 mM 1 mM Merck, 40 mg
tablets % viability by ~100 ~100 AlamarBlue test Methotrexate, 1 mM
10 mM 1 mM 10 mM 1 mM 10 mM Sigma cat. no. M-9929 % viability by
~100 ~90 AlamarBlue test Tacrine, 25 uM 250 uM 25 uM 250 uM 25 uM
250 uM Sigma cat. no. A-3773 % viability by ~100 ~75-80 AlamarBlue
test Valproate, 0.4 mg/ml 4 mg/ml 0.4 mg/ml 4 mg/ml 0.4 mg/ml 4
mg/ml Sigma cat. no. P-4543 % viability by ~100 ~95 AlamarBlue test
Notes: 1. Each compound was dissolved in HIM cell culture medium
(In Vitro Technologies) containing 0.2% DMSO (Sigma cat. no.
D-5879). 2. The AlamarBlue assay was performed only at the 24-hr
time point following exposure to the toxin of interest. A
corresponding vehicle control (0.2% DMSO) sample was also isolated
at 3, 6, and 24-hr time points for each toxin.
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090197258A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
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An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090197258A1).
An electronic copy of the "Sequence Listing" will also be available
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* * * * *
References