Microarrays Reveal Breadth of Toxicity
Benzene is both widely used and widely studied. Yet, although the chemical
is strongly associated with leukemia in humans, questions remain regarding its
mechanism of action. Hoping to better understand the genetic mechanisms behind
benzene's hematotoxicity and leukemogenicity, a group of researchers from Japan
and Korea used cDNA microarrays to analyze mouse bone marrow tissue both during
and after a two-week exposure to the compound by inhalation [EHP 111:1411-1420]. The researchers found, among other discoveries,
that benzene may perturb cell cycling that is mediated by the gene for the protein
p53, triggering a host of fatal problems at the cellular level and thus causing
blood cell malignancies epigenetically (that is, without encoding the information
in the genetic code).
A
bad actor in blood. New research shows that benzene's toxic effects--including
leukemogenicity and hematotoxicity--are wrought through many pathways. Thus,
multiple genes may be implicated.
image credit: Photodisc, Christopher G. Reuther/EHP |
Benzene is used in fuels, as an industrial solvent, and in other manufacturing
applications, and is also found in cigarette smoke. Human populations generally
are exposed through polluted ambient air or contaminated water. Benzene is known
to cause hematotoxicity and blood tumors in humans and mice. Studies so far
have focused on benzene's carcinogenic and genotoxic metabolites, which cause
various types of tumors in a number of mouse organ systems. Hepatic enzymes
convert inhaled benzene into genotoxic metabolites. Then, to add insult to injury,
a number of these benzene metabolites (primarily phenol, hydroquinone, catechol,
and trans-trans muconic acid) actually intensify the chemical's toxic
effect on an organ.
Past studies have suggested that benzene's toxic effects on bone marrow tissue--its
major target organ--may be enacted through multiple pathways, including growth
factor regulation, oxidative stress reduction, DNA damage repair, cell cycle
regulation, and apoptosis. Also, genetic variations may upset the cellular-environmental
homeostasis that protects bone marrow cells from toxic effects such as those
caused by benzene, resulting in altered gene expression. Therefore, the authors
suggest, studying just a few specific genes may not be enough to thoroughly
explain the complex molecular mechanisms of benzene-induced hematotoxicity and
leukemogenicity.
With this in mind, the research team conducted broad cDNA microarray analyses
using multiple gene expression profiling technologies. The team analyzed mouse
bone marrow tissue during and after exposure to 300 parts per million benzene
over a 2-week period for 6 hours a day, 5 days a week. Two types of C57BL/6
mice were used--standard wild-type mice possessing the gene for p53 and p53-knockout
mice. The mice were randomly grouped into control and benzene-exposed groups.
Twice during the exposure period and then 3 days after the full 2-week exposure,
the researchers collected bone marrow from both femurs of each mouse in each
group. RNA was extracted from this tissue and used to synthesize cDNA, which
was then hybridized onto a microarray chip. The resulting array of gene fragments
was scanned as a digital image and analyzed using software that searched for
clustering genes specifically expressed and/or suppressed in each group.
The researchers found that benzene caused DNA damage in cells during all phases
of the cell cycle. In the benzene-exposed wild-type mice, DNA repair genes were
activated, but they were suppressed in the p53-knockout mice. Mice in the latter
group were therefore susceptible to benzene's direct genotoxic leukemogenicity,
whereas those in the former still experienced epigenetic leukemogenicity via
cell-cycle perturbations despite DNA repair.
Besides the p53-mediated pathway, the investigators identified other specific
genes that may be involved in G1 cell cycle arrest and apoptosis following benzene
exposure, and confirmed that certain repair genes--including the tuberous sclerosis
gene and the metallothionein 1 gene--are also triggered by such exposure. They
also found that, during benzene exposure, the production of blood cells was
arrested due to alterations in the expression of cell cycle checkpoint genes
in the wild-type mice. However, production continued in the p53-knockout mice,
an important difference that the researchers say could point to mechanisms of
benzene's hematotoxicity.
The researchers' cDNA microarray analyses supported the theory that the gene
for p53 mediates the effect of benzene on bone marrow tissue by regulating specific
genes instrumental in cell cycle arrest, apoptosis, and DNA repair. Because
careful simultaneous screening of different expression patterns of many interrelated
genes between the two groups is necessary, the researchers write, toxicogenomics
should prove extremely useful for future investigations into the toxicity and
leukemogenicity mechanisms of benzene.
Jennifer Medlin
Effect of SNPs on OPs
Age and Race Variations Explored
Newborns produce substantially less of the enzyme paraoxonase-1 (PON1)--which
detoxifies organophosphate pesticides--than do adults, potentially leaving them
more vulnerable to organophosphate exposures. Genetic differences in PON1 activity
are also more pronounced in newborns than in adults, according to recent research
by Jia Chen and colleagues at the Mount Sinai School of Medicine [EHP 111:1403-1409]. By helping to determine which groups are most
susceptible based on age and genetic factors, these results may have implications
for setting exposure standards.
Metabolites of organophosphates damage the nervous systems of insects and
humans by reducing the ability of the enzyme cholinesterase to regulate the
electrochemical signals between neurons. When cholinesterase levels drop, neurons
become overstimulated and send repeat signals that can eventually cause muscle
weakness, paralysis, and death. PON1 breaks down organophosphates before they
can cause nerve damage.
The current study is part of ongoing research on the neurodevelopmental risks
posed by exposure to the organophosphate pesticide chlorpyrifos among an inner-city
population in New York City. Once among the most commonly used insecticides,
chlorpyrifos was banned in 2000 for many residential uses in the United States
because of concern over children's health. Like other organophosphates, chlorpyrifos
and its toxic metabolite chlorpyrifos oxon can cross the placenta. Therefore,
exposures by pregnant women can affect their unborn children. However, little
is known about the effects of low-level exposures on children's development,
including their ability to learn later in life. Chlorpyrifos is still approved
for many agricultural uses, and children in rural areas continue to be exposed.
Researchers have identified five single-nucleotide polymorphisms that affect
PON1 production, three in the promoter region of the PON1 gene (-909,
-162, -108) and two in the coding region (L55M, Q192R). PON1 has also been linked to, or tends to be inherited along with, two other genes,
PON2 and PON3.
Separate
at birth. New data reveal racial differences in PON1 activity in newborns.
This means some infants may be more vulnerable than others to the effects
of organophosphates, which are broken down by this enzyme.
image credit: Eyewire |
From March 1998 through March 2002, the Mount Sinai researchers genotyped and
measured PON1 activity in the blood of an ethnically diverse group of 402 expectant
mothers and 229 newborns. Participants identified themselves, or were identified
by their parents, as Caucasian (82 mothers, 56 newborns), African American (117
mothers, 66 newborns), or Caribbean Hispanic (203 mothers, 107 newborns). Blood
samples were genotyped for the five PON1 polymorphisms. Because the researchers
are also studying the linkage of PON genes, samples were also genotyped
for a common PON2 polymorphism (C311S). The level of PON1 activity in
each blood sample was determined by an assay that measures hydrolysis of phenylacetate.
As expected, the results showed that PON1 activity of newborns was less than
that of adults, and so newborns are potentially more susceptible to the effects
of chlorpyrifos exposure. In addition, the Mount Sinai researchers found that
some groups of newborns may be more vulnerable than others. The presence of
PON1 polymorphisms and PON1 activity varied among racial/ethnic groups.
PON1 activity in the blood of the expectant mothers was 4.6, 3.6, and 2.6 times
greater than in that of newborns for Caucasians, Caribbean Hispanics, and African
Americans, respectively. In addition, the impact of genetic variability was
greater in the newborns than in the adults. None of the polymorphisms affected
PON1 activity in the women by more than 35%. However, among the newborns, several
of the polymorphisms affected PON1 activity by as much as 200%. For example,
Caucasian infants with the CC polymorphism of the -108 PON1 promoter had, on average, more than twice the PON1 activity of infants with
the TT polymorphism.
The researchers further found that the polymorphisms tended to be inherited
in predictable patterns, a phenomenon referred to as "linkage disequilibrium."
There was significant linkage disequilibrium among the three promoter polymorphisms,
and among the promoter polymorphisms and the coding polymorphism L55M. These
relationships were strongest for Caucasian participants and weakest for African-American
participants. In addition, there was significant linkage disequilibrium among
the PON1 promoter polymorphisms and the PON2 polymorphism C311S.
These results may contribute to a better understanding of rates of recombination
in genes, as well as provide a basis for future epidemiological studies.
Kris Freeman