TOXICOGENOMICS

[Guest guest]

Yet still who has decided what exact human code is minus genetic

mutation/alteration from environmental factors absorbed?....

this " NEW' field of " better living " ?.....as opposed to spewing it out

everywhere, dispensing it everywhere and poisoning all of us

first......phasing it out so no monies are lost by the poisoners for profit

and still being applied by stockpilers on the QT..and finally banning it

after it is environmentally laden and poisoning us daily for the next >40+

years with

Onus on the chemical company and EPA confidentiality protection still the

order of the day to produce false data.....

DMREILLY

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Better Living Through Toxicogenomics?

Gene expression profiling on " tox chips " promises to speed toxicity analyses,

streamline drug development, and improve consumer safety.

By Lane & Pray

J. Viola

http://www.the-scientist.com/yr2002/mar/profile1_020304.html

Poison in Your Pantry? A new field helps scientists measure the toxicity of

nearly 80,000 chemicals used in consumer goods today.

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Toxicologists traditionally use animals to test the toxicity of chemicals and

other substances. But the brand new field of toxicogenomics, which applies a

whole-genome approach to toxicology questions, is changing all that. Still in

its infancy, this field is destined to change the way toxicologists think and

act, and could even help optimize the drug-development process. " It holds

great promise for the future, " says Jay Goodman, a Michigan State University

toxicologist. " Toxicogenomics is a tool that can improve the assessment of

potential toxicity. " Phil Iannaccone, a researcher at Northwestern University

Medical School and invited author of a recent Environmental Health Perspective

editorial on the new technology,1 agrees: " The hope is that the observed

patterns will be characteristic of a class of toxicants, such as polycyclic

aromatic hydrocarbons versus peroxysome proliferators. Eventually one might

hope for specificity allowing actual identification of the chemical, " he

says. " For now it is exciting enough that one might be able to determine if

an unknown chemical is likely to behave as a certain class of toxicants or

not. "

The new field is based on the premise that tumors, disease, and other

physical responses to toxic chemicals find their origins in gene expression,

which depends on the environment—chemical or otherwise. Determining how genes

respond to a toxicant, then, could be a direct measure of toxicity. It's

faster, cheaper, and more accurate than animal testing, which often takes

years to perform; examining gene expression takes only days or months.

That's welcome news, as there are over 80,000 substances being used in

commerce such as drugs, food additives, cosmetics, and chemicals, but only a

fraction has been thoroughly tested for toxicity, says McClure,

Chief, Organs and Systems Toxicology Branch, Office of Program Development,

National Institute of Environmental Health Sciences (NIEHS). Toxicogenomics

could accelerate the speed at which these substances are tested.

Microarrays Fit the Bill

The key toxicogenomicist's tool is the microarray, which allows scientists to

simultaneously assess the expression of thousands of genes and produce a

profile, or signature, for each toxin. Like traditional biomarkers, such as

blood enzyme levels, microarray profiles can flag substances as hazardous,

but this new biomarker provides greater precision in identifying hazards.

Each chip's probe set is manufacturer-dependent (see sidebar), but they

typically include genes implicated in DNA replication and repair, and

apoptosis; transcription factors; signaling molecules; and genes known to

respond to various cellular insults. Toxicologists use these microarrays in

several ways. Instead of groping in the dark for a few genes that might be

involved in the toxicant response, scientists can use toxicogenomics to get a

complete picture of all affected genes, and then focus on the promising

candidates. This knowledge provides clues as to why animals develop cancer,

liver damage, heart problems, or birth defects in response to a toxin.

Researchers also can screen an environment for toxins by assessing the gene

expression characteristics of organisms living in that environment. And they

can measure the gene expression profiles of clinical trial participants to

determine a drug's toxicity, its effects on the body, and the effects of

different dosages.

These applications could change the way pharmaceutical companies operate.

Drug discovery methods such as combinatorial chemistry have greatly increased

the number of drug leads, but taking one compound through the development

process often costs millions of dollars. If, during clinical trials, that

compound is found to be toxic, the money would be wasted. Pharmaceutical

companies can reduce R & D costs by running the compound through microarray

testing early on in the process.

A Complement, Not a Replacement

The NIEHS established the National Center for Toxicogenomics (NCT) in

September 2000. According to the Center's mission statement, its goal is " to

use the methodologies and information of genomics science to significantly

improve our understanding of basic biological responses to environmental

stressors/toxicants. " NCT director Tennant explains that

toxicogenomics fundamentally changes the way toxicologists carry out their

work: " In football you try to identify the person carrying the ball and

tackle him. With global gene expression, you can tackle the entire football

team at one time and throw out the players until you find the ball. "

Of course, it helps to have the entire team on one chip. With all kinds of

genes assembled on one array, probes can exclude those genes that don't play

a role in the toxic response. But knowing which genes change their expression

in the presence of toxicants is of only limited value. Scientists must be

able to correlate these changes with something tangible, such as a tumor.

Otherwise, the fact that certain genes become up- or down-regulated doesn't

mean much.

" If you take a pesticide and throw it at a cell culture, you're going to get

a response, but you don't know what the significance of the response is, "

says Spencer, director, Center for Research on Occupational and

Environmental Toxicology at Oregon Health Sciences University. Goodman adds,

" In order to make progress, this new tool must be linked to basic principles

of toxicology—for example, dose- and time-response relationships—and we need

to understand that a simple change in gene expression is not necessarily

indicative of toxicity. "

So, researchers will continue to perform animal testing, while toxicologists

anchor gene expression profiles to actual phenotypic effects. Starting with

chemicals already known to cause cancer, a gene expression profile could be

correlated with carcinogenicity. Toxicologists could then compare the

signature of an unknown chemical to that of known toxicants. A matching

signature then offers clues to that compound's effects.

Bioassays that don't use animals can also help interpret gene expression

profiles, measure toxicant-induced DNA damage, and can determine

neurotoxicity, immunotoxicity, reproductive and developmental toxicology, and

genetic toxicology. The unknown chemical's signature can help the researcher

determine if the toxicant induces a biological response. Then, the most

relevant bioassay can be selected. This makes sense, as some bioassays can be

extremely expensive and time consuming, such as the rodent cancer bioassay,

which requires four years, 1,200 animals, and millions of dollars to execute

and analyze.2

Courtesy of PHASE-1 Molecular Toxicology Inc.

An Eye Towards the Future: Gene expression profiling may one day replace

traditional bioassays, but not until scientists can correlate the data with a

tangible, physical effect.

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Microarrays offer several advantages, despite their reliance on bioassays for

data validation. The approach could enhance a bioassay's sensitivity and

interpretability, possibly reducing its cost. In addition, because low doses

of chemicals can affect gene expression, bioassays can be performed using

less of the potential toxicant. This produces a more realistic picture of how

humans might be affected by the chemicals because everyday exposures normally

occur in small doses. Microarray testing also allows scientists to observe

how humans react to a toxicant rather than having to extrapolate data from

rats, mice or other animals.

Scientists can also observe how acute exposure to a chemical might differ

from chronic exposure. In a recent study, researchers in England exposed

human hepatoma cell line HepG2 to two hepatotoxins, carbon tetrachloride and

ethanol, and collected gene expression data using microarrays.3 After two

hours, the scientists found that gene expression hadn't changed much, but

after 24 hours, carbon tetrachloride caused an increase in expression of

genes involved in extracellular transport and cell signaling, whereas ethanol

exposure caused a decrease in the expression of genes involved in the stress

response and metabolism.

A Sea of Data

Analyzing these results is not easy. Microarrays generate giant lists of

numbers representing changes in gene expression. " It's one thing to run an

array. That's the easy part, " Tennant says. " Coming to understand what those

data are telling you about is a substantial effort. " That's where

bioinformatics comes in, says McClure. Bioinformaticians can help

toxicologists make meaningful conclusions out of the deluge of data points.

But the trial of wading through the data is nothing compared to the effort it

took to study how toxicants affected genes before toxicogenomics came along.

At that time, it was, " One gene, one protein at a time, " observes Leona Samson

, a toxicologist at Massachusetts Institute of Technology. Now, " our eyes are

being opened to a multitude of responses that are helpful to the cell for

recovery. "

Using software to archive, compare, and interpret microarray data,

researchers can now begin the task of compiling databases against which new

compounds can be tested. For example, Santa Fe, NM-based PHASE-1 Molecular

Toxicology Inc., is developing a database that will hold the gene expression

profiles of a whole spectrum of chemicals.

The NCT is also building a database. In going forward with that task, the NCT

announced in November the formation of the Toxicogenomics Research

Consortium. Organized by an NIEHS team led by McClure and Ben Van Houten, the

Consortium is a $37 million effort.

To make a useful database, which can serve as a universal reference, the

researchers must establish standards, fine tuning environmental factors and

experimental conditions. They'll have to consider dosages, and finely control

how they culture cells and treat animals—including such details as lighting,

nutrition and feeding schedules, and handling effects. This will ensure that

any changes in gene expression result from the tested chemical and not from

the ambient environment.

In the future, toxicogenomics will likely play an important role in

personalized medicine, says Jongedijk, marketing and sales director at

PHASE-1's Belgium headquarters. Yet despite its promise, very few companies

offer arrays specifically for toxicological applications, and fewer still

have developed technology that's affordable and accessible to individual

academic researchers.

Toxicogenomics is big-ticket science; most of the players are pharmaceutical

and biotech companies. But, as Iannaccone points out, " if you have access to

a biotech center, which most universities have now, then the individual

investigator absolutely can do this. Most of the interesting work in the past

few years can be traced to a single postdoc in the lab that published the

work. Generally this person picked this up as a project without prior

experience. They then wind up getting job offers in drug companies and are

whisked out of the lab! "

Lane (lanelaura@...) is a freelance writer in San Francisco.

Pray (lpray@...) is a freelance writer in Leverett, Mass.

References

1. P.M. Iannaccone, " Toxicogenomics: 'The call of the wild chip,' "

Environmental Health Perspectives, 109:A8-11, January 2001.

2. E.F. Nuwaysir et al., " Microarrays and toxicology: The advent of

toxicogenomics, " Molecular Carcinogenesis, 24[3]:153-9, 1999.

3. H.M. Harries et al., " The use of genomics technology to investigate gene

expression changes in cultured human liver cells, " Toxicology In Vitro,

15[4-5]:399-405, August-October 2001.

Chip Makers

IN FOCUS | Lane & Pray

ToxChip1 was the prototype toxicology chip, says Afshari, one of the

chip's developers at the National Institute of Environmental Health Sciences

(NIEHS) Microarray Center in Research Triangle Park, NC (www.niehs.nih.gov/nct

). In developing the chip, Afshari and colleagues consulted with experts to

identify toxicologically relevant genes; the expertise they rallied in

developing the chip is one of ToxChip's unique features, says

Afshari.Initially comprised of 2,000 genes, the chip now holds 12,000

genes—including 6,000 unknown, which is more than any of the commercially

available toxicology-targeted microarrays. For academicians, the more genes

examined, the better, says Afshari, because it is impossible to predict all

toxicologically relevant genes. Also, not every gene that seems important

based on past research will necessarily be informative in an expression-based

array. RNA from exposed animals and humans is a very valuable resource,

observes Afshari, and researchers want to get as much information from it as

they can.

The ToxChip isn't for sale, though the gene list is free and accessible on

the Internet. Researchers can, however, collaborate with NIEHS after

submitting a proposal for in-house review (dir.niehs.nih.gov/microarray/).

Currently, NIEHS has over 50 microarray-based collaborations in both basic

and toxicology-based studies. The group hopes that by making the gene list

public, other researchers will alert them to important genes that should be

added to the ToxChip, so that its usefulness will continue to evolve.

For commercial toxicogenomics applications, companies are required to obtain

a license from Discovery Partners International Inc. (DPII) of San Diego.

DPII's subsidiary, Xenometrix, owns and has exclusive rights to US patent

5,811,231, " Methods and Kits for Eukaryotic Gene Profiling, " issued in 1998.

According to Xenometrix president ine Gee, the patent is fundamental to

toxicogenomics. It covers " the creation of many multi-gene expression

profiles resulting from exposure of eukaryotic cells ... to pharmaceutical

compounds and other chemical entities, " according to a press release

announcing the patent's issuance. Several companies offer " tox chips "

commercially.

Santa Clara, Calif.-based Affymetrix Inc. (www.affymetrix.com) introduced its

Rat Toxicology U34 Array, containing over 850 genes and ESTs, in 1999.

Gaithersburg, Md.-based Gene Logic (www.genelogic.com) used this chip, plus

the Human Genome U95 array, to develop a reference database, the GeneExpress®

ToxExpressâ„¢ Module. ToxExpress is a " reference set of sentinel genes and

their gene expression patterns that are predictive " of toxicity, says Bob

Burrows, Gene Logic's corporate communications director. The data are based

on profiling selected pharmaceuticals in rats and human cell lines, at both

therapeutic and toxic doses, to examine gene expression changes following

exposure to a toxic compound.

Companies that already have their own in-house toxicogenomics programs can

use ToxExpress as a " predictive backdrop, or library, of classic toxicity

patterns, " says Burrows. Currently, the company has 10 pharmaceutical and

biotech subscribers.

Palo Alto, Calif.-based CLONTECH Laboratories (www.clontech.com) offers the

Atlasâ„¢ Human, Mouse, and Rat Toxicology 1.2 nylon-based arrays. Each contains

over 1,000 genes selected by scientists at the Environmental Protection

Agency (EPA) for their involvement in cellular responses to toxicant

exposure.

Will Scovill, Atlasâ„¢ product manager, says CLONTECH's products are accessible

to individual researchers because they are less expensive than similar

products on the market and do not require any specialized techniques or

equipment. The only extra hardware that the user needs for a complete

Atlas-based toxicogenomic study is a phosphorimager to read the hybridization

profile.

CLONTECH's arrays contain a relatively small number of genes, but each has

established importance in the toxicology field, says Scovill, which allows

researchers to focus on the most relevant genes. " It makes the bioinformatics

end of toxicogenomics much easier, " he adds. Users receive free access to the

AtlasInfoâ„¢ Bioinformatics Database, which includes information such as

alternative names and known functions for each gene, journal citations, and

links to related databases.

One of Santa Fe, NM-based PHASE-1 Molecular Toxicology's (www.phase1tox.com)

primary functions is to provide pre-clinical toxicogenomic services to

biotech and pharmaceutical companies, with a flexible, customized approach.

Smaller companies often submit their samples and say " we want you to do

everything, " says Jongedijk, marketing and sales director at PHASE-1's

Belgium headquarters. But larger companies sometimes prefer to do their own

work, in which case they buy PHASE-1's microarrays and access to its database

and software, but perform their own analyses.

PHASE-1 produces the Rat 700, Canine 140, and Custom arrays. Like CLONTECH's

arrays, these chips contain far fewer probes than the ToxChip, but from a

regulatory perspective, it may make more sense to use the smaller chips, says

Afshari. The more genes there are, the larger the data set, and the more

unexplained data. This can cause trouble with the FDA, because the agency

requires that every data point be explained.

Most of the genes on PHASE-1's arrays are picked using empirical validation,

says Jongedijk, unlike other toxicology arrays on the market, which are

knowledge-based. In other words, the gene sets have been identified

experimentally by PHASE-1 scientists, and not culled from peer-reviewed

literature.

Expression profile data are analyzed using PHASE-1 software and compared to

gene expression patterns of known compounds in PHASE-1's TOXbankâ„¢ database,

and to small sets of proprietary organ-specific toxicity biomarkers. TOXbank

was developed from thousands of experiments on the effects of over 150

compounds in rats. " All data are generated in-house in a very strict

protocol, " says Jongedijk. Because they don't use external data, the

company's reference database is not confounded by the kind of variation

expected in databases made up of multiple sources, he says.

PHASE-1 also produces software that can generate, store, and interpret the

mounds of data that arise from molecular toxicology experiments. One of the

programs, called MATRIXexpressâ„¢, enables the manipulation and extraction of

relevant data from the company's toxicology profile database, including

sorting and analysis, annotation, and clustering analysis to enhance the

predictive value of the inherent data.

1. E.F. Nuwaysir, " Microarrays and toxicology: The advent of toxicogenomics, "

Molecular Carcinogenesis, 24:153-9, 1999.

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The Scientist 16[5]:37, Mar. 4, 2002