Daphnia magna ecotoxicogenomics provides mechanistic insights into metal toxicity.
ABSTRACT Toxicogenomics has provided innovative approaches to chemical screening, risk assessment, and predictive toxicology. If applied to ecotoxicology, genomics tools could greatly enhance the ability to understand the modes of toxicity in environmentally relevant organisms. Daphnia magna, a small aquatic crustacean, is considered a "keystone" species in ecological food webs and is an indicator species for toxicant exposure. Our objective was to demonstrate the potential utility of gene expression profiling in ecotoxicology by identifying novel biomarkers and uncovering potential modes of action in D. magna. Using a custom D. magna cDNA microarray, we identified distinct expression profiles in response to sublethal copper, cadmium, and zinc exposures and discovered specific biomarkers of exposure including two probable metallothioneins, and a ferritin mRNA with a functional IRE. The gene expression patterns support known mechanisms of metal toxicity and reveal novel modes of action including zinc inhibition of chitinase activity. By integrating gene expression profiling into an environmentally important organism, this study provides experimental support for the utility of ecotoxicogenomics.
- SourceAvailable from: berkeley.edu[show abstract] [hide abstract]
ABSTRACT: The availability of genome-scale DNA sequence information and reagents has radically altered life-science research. This revolution has led to the development of a new scientific subdiscipline derived from a combination of the fields of toxicology and genomics. This subdiscipline, termed toxicogenomics, is concerned with the identification of potential human and environmental toxicants, and their putative mechanisms of action, through the use of genomics resources. One such resource is DNA microarrays or "chips," which allow the monitoring of the expression levels of thousands of genes simultaneously. Here we propose a general method by which gene expression, as measured by cDNA microarrays, can be used as a highly sensitive and informative marker for toxicity. Our purpose is to acquaint the reader with the development and current state of microarray technology and to present our view of the usefulness of microarrays to the field of toxicology.Molecular Carcinogenesis 04/1999; 24(3):153-9. · 4.27 Impact Factor
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ABSTRACT: Microarrays have the potential to significantly impact our ability to identify toxic hazards by the identification of mechanistically relevant markers of toxicity. To be useful for risk assessment, however, microarray data must be challenged to determine reliability and interlaboratory reproducibility. As part of a series of studies conducted by the International Life Sciences Institute Health and Environmental Science Institute Technical Committee on the Application of Genomics to Mechanism-Based Risk Assessment, the biological response in rats to the hepatotoxin clofibrate was investigated. Animals were treated with high (250 mg/kg/day) or low (25 mg/kg/day) doses for 1, 3, or 7 days in two laboratories. Clinical chemistry parameters were measured, livers removed for histopathological assessment, and gene expression analysis was conducted using cDNA arrays. Expression changes in genes involved in fatty acid metabolism (e.g., acyl-CoA oxidase), cell proliferation (e.g., topoisomerase II-Alpha), and fatty acid oxidation (e.g., cytochrome P450 4A1), consistent with the mechanism of clofibrate hepatotoxicity, were detected. Observed differences in gene expression levels correlated with the level of biological response induced in the two in vivo studies. Generally, there was a high level of concordance between the gene expression profiles generated from pooled and individual RNA samples. Quantitative real-time polymerase chain reaction was used to confirm modulations for a number of peroxisome proliferator marker genes. Though the results indicate some variability in the quantitative nature of the microarray data, this appears due largely to differences in experimental and data analysis procedures used within each laboratory. In summary, this study demonstrates the potential for gene expression profiling to identify toxic hazards by the identification of mechanistically relevant markers of toxicity.Environmental Health Perspectives 04/2004; 112(4):428-38. · 7.26 Impact Factor
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ABSTRACT: Co-contamination with complex mixtures of carcinogenic metals, such as chromium, and polycyclic aromatic hydrocarbons is a common environmental problem with multiple biological consequences. Chromium exposure alters inducible gene expression, forms chromium-DNA adducts and chromium-DNA cross-links, and disrupts transcriptional activator-co-activator complexes. We have shown previously that exposure of mouse hepatoma Hepa-1 cells to chromate inhibits the induction of the Cyp1a1 and Nqo1 genes by dioxin. Here we have tested the hypothesis that chromium blocks gene expression by interfering with the assembly of productive transcriptional complexes at the promoter of inducible genes. To this end, we have studied the effects of chromium on the expression of genes induced by benzo[a]pyrene (B[a]P), another aryl hydrocarbon receptor agonist, and characterized the disruption of Cyp1a1 transcriptional induction by chromium. Gene expression profiling by using high density microarray analysis revealed that the inhibitory effect of chromium on B[a]P-dependent gene induction was generalized, affecting the induction of over 50 different genes involved in a variety of signaling transduction pathways. The inhibitory effect of chromium on Cyp1a1 transcription was found to depend on the presence of promoter-proximal sequences and not on the cis-acting enhancer sequences that bind the aryl hydrocarbon receptor-aryl hydrocarbon receptor nuclear translocator complex. By using transient reporter assays and chromatin immunoprecipitation analyses, we found that chromium prevented the B[a]P-dependent release of HDAC-1 from Cyp1a1 chromatin and blocked p300 recruitment. These results provide a mechanistic explanation for the observation that chromium inhibits inducible but not constitutive gene expression.Journal of Biological Chemistry 03/2004; 279(6):4110-9. · 4.65 Impact Factor
Mechanistic Insights into Metal
H E L E N C . P O Y N T O N ,†J U L I A R .
V A R S H A V S K Y ,†B O N N I E C H A N G ,†
G I O R G I O C A V I G I O L I O ,‡S A R A H C H A N ,†
P A T R I C I A S . H O L M A N ,†A L E X A N D R E V .
L O G U I N O V ,†D A R R E N J . B A U E R ,§K E L L Y
K O M A C H I ,⊥E L I Z A B E T H C . T H E I L ,‡
E D W A R D J . P E R K I N S ,#O W E N H U G H E S ,⊥
A N D C H R I S D . V U L P E *, †
Nutritional Sciences and Toxicology, University of California,
Berkeley, California 94720, Center for BioIron at CHORI
(Children’s Hospital Oakland Research Institute),
Oakland, California 94609, Hubbard Center for
Genome Studies, University of New Hampshire,
Durham, New Hampshire 03824, Eon/Terragenomics,
Davis, California 95616, and Environmental Laboratory,
U.S. Army Engineer Research and Development Center,
Vicksburg, Mississippi 39180
Toxicogenomics has provided innovative approaches to
chemical screening, risk assessment, and predictive
toxicology. If applied to ecotoxicology, genomics tools
could greatly enhance the ability to understand the
modes of toxicity in environmentally relevant organisms.
Daphnia magna, a small aquatic crustacean, is considered
a “keystone” species in ecological food webs and is
an indicator species for toxicant exposure. Our
objective was to demonstrate the potential utility of
gene expression profiling in ecotoxicology by
identifying novel biomarkers and uncovering potential
modes of action in D. magna. Using a custom D. magna
cDNA microarray, we identified distinct expression
profiles in response to sublethal copper, cadmium, and
zinc exposures and discovered specific biomarkers of
exposure including two probable metallothioneins, and a
ferritin mRNA with a functional IRE. The gene expression
patterns support known mechanisms of metal toxicity
and reveal novel modes of action including zinc inhibition
of chitinase activity. By integrating gene expression
profiling into an environmentally important organism, this
study provides experimental support for the utility of
From evolutionary biology to the medical sciences, almost
every field of biology has benefited from the genomics
genomics into toxicological studies. In 1999, Nuwaysir et al.
described how microarray technologies could transform
toxicology, presenting the possibilities of a new field called
been used to identify and confirm mechanisms of action of
(3), and identify novel biomarkers of exposure including
exposure to copper (4) and other metals (5). Perhaps the
greatest potential for microarrays in toxicology is the ability
for such a classification system (6), and databases such as
Chemical Effects in Biological Systems (CEBS) are presently
being developed to store large amounts of gene expression
data to make this a reality (7).
The accomplishments of toxicogenomics, if applied to
ecotoxicology, could facilitate the basic tasks of monitoring
contaminant levels, identifying the chemicals responsible
for toxicity in impaired waters, and helping protect ecosys-
tems and human health by encouraging the design of less
harmful chemicals (8). A few studies have shown how
genomic technologies could be employed in ecotoxicology
magna cDNA microarray enriched in transcripts related to
illustrated the usefulness of custom-made microarrays for
studying gene expression in a nontraditional organism.
In the present study, we continue to build on the proof-
the development of a cDNA microarray for D. magna (water
flea), a standard test organism for freshwater ecotoxicity
studies (11). We identified distinct gene expression profiles
for three metal exposures: copper, cadmium, and zinc at
sublethal concentrations. Moreover, we have discovered
metallothioneins, and homologues to glutathione-S-trans-
us to uncover unknown modes of toxicity, including a
during zinc exposure. Further investigation confirmed that
Zn causes a decrease in chitinase activity, linking the gene
expression changes to a physiological response. This study
illustrates the power of genomics to go beyond a focus on
individual genes by establishing expression profiles of
contribute to the toxicity of these three metals.
Materials and Methods
Maintenance of D. magna Cultures. Genetically homoge-
neous D. magna, purchased from Aquatic Research Organ-
water hardness (the constituents of COMBO media are
described in ref 12) and maintained at 23.5 °C in a Percival
hardness, and alkalinity were measured, recorded, and
reported in Table S2 (Supporting Information).
Acute and Chronic Toxicity Assays. Acute and chronic
USEPA Whole Effluent Toxicity (WET) protocol (11) and
USEPA chronic toxicity WET protocol (13). First instar D.
magna were placed in 25 mL of media containing varying
* Corresponding author phone: (510) 642-1834; fax: (510) 642-
0535; e-mail: email@example.com.
†University of California.
‡Center for BioIron at CHORI.
§University of New Hampshire.
#U.S. Army Engineer Research and Development Center.
Environ. Sci. Technol. 2007, 41, 1044-1050
10449ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 3, 2007 10.1021/es0615573 CCC: $37.00
2007 American Chemical Society
Published on Web 12/20/2006
NH), cadmium sulfate (Fisher Scientific), or zinc chloride
(Sigma-Aldrich, St. Louis, MO). Eight concentrations were
for the acute test and reproduction for the chronic test. The
LC50, EC50, and NOEC were determined using the statistical
procedures outlined by the USEPA (11).
library (generous gift from D. Bauer at University of New
Hampshire, NH, and J. Colbourne at Indiana University, IN)
were PCR amplified from the pDNR-LIB vector using the
following primers: forward, AGTCGACGGTACCGGACATA,
and reverse, GCCAAACGAATGGTCTAGAAA. PCR products
were purified by ethanol precipitation and resuspended in
distilled water. Clones were checked by electrophoresis on
a 1.2% agarose gel to ensure uniform PCR products. cDNA
clones were printed onto lysine-coated glass slides by the
UCB Nutritional Sciences and Toxicology genomics facility.
Chemical Exposures. Chemical exposures were per-
formed using ∼40 adult (16-18 day old) D. magna placed
in 2 L of COMBO media for 24 h. For microarray hybridiza-
tions, we exposed D. magna to a sublethal concentration of
(see Table S1, Supporting Information) for each metal. A
zero concentration control was performed alongside the
metal exposures. For the chitinase enzyme assay, D. magna
were exposed to the 1/10 LC50 for each metal. Additional
microarray experiments, three biological replicates were
replicate exposure was performed for real-time PCR con-
firmation, and three separate exposures were done for the
chitinase enzyme assays. Following each 24-h exposure, D.
hybridizations and real-time PCR or crude protein was
isolated for chitinase activity studies.
RNA Isolation. D. magna were harvested by gently
removing each daphnid from the culture and immediately
grinding them in liquid nitrogen using a pestle and mortar.
(Invitrogen, Carlsbad, CA).
Microarray Hybridization. Before proceeding to reverse
transcription, RNA from both the unexposed and exposed
D. magna was split into two pools, to provide two replicate
hybridizations for each metal exposure. cDNA was synthe-
from total RNA in the presence of aminoallyl-labeled dUTP.
Fluorescence labeling proceeded by incubating the ami-
noallyl-labeled cDNA with Cy5 or Cy3 fluorescent dyes
(Amersham Biosciences, Piscataway, NJ). The dyes were
was labeled with Cy3 in one hybridization and Cy5 in the
other. The labeled cDNA pools from the unexposed and
to two different microarrays, there were six hybridizations
for each metal. Scanning and quantification was performed
using an arrayWoRx Biochip Reader (Applied Precision,
Issaquah, WA) and GenePix software version 3.01 (Axon
Instruments, Union City, CA). Detailed information about
Omnibus (GEO) (located at http://www.ncbi.nlm.nih.gov/
geo) with the accession number GSE4759.
Identification of Candidate Differentially Expressed
Genes. The statistical methods used to normalize the data
and identify differentially expressed genes are described in
normalized to remove possible nonlinearity, if any, and
checked for homogeneity using box plots. As an alternative
to between-slide normalization, we applied an approach
based on sequential single-slide data analysis and utilized
to identify differentially expressed cDNAs. Our algorithms
are implemented as software written in S-plus language (R
version of the software is available in Louginov et al. (14)).
The software used microarray data from single-slide experi-
ments as an input and generated tables with candidate
differentially expressed cDNAs, different types of the cor-
responding ratios, unadjusted p-values, adjusted p-values,
and q-values. We applied an average false positive cutoff of
1 to identify candidates for differential gene expression.
cDNAs differentially expressed in both technical replicates,
and in two of the three, biological replicates were chosen as
candidate differentially expressed cDNAs.
Sequencing of Differentially Expressed cDNAs. The
plasmids containing the cDNAs determined to be differen-
Identification of Protein Homologues and Prediction
of Protein Function. Translated BLAST searches (tblastx)
to the sequenced cDNA clones (http://greengene.uml.edu/
Batch.html). cDNAs without homology to known proteins
and cDNAs whose closest protein homologue was unchar-
acterized were further analyzed for possible protein func-
tion. cDNAs were translated using Expasy translation tool
(http://us.expasy.org/tools/dna.html), and all possible pro-
tein fragments were characterized using PredictProtein
Real-Time PCR. To confirm the differential expression,
isolated following 1/10 LC50 metal exposures was reverse
transcribed using Superscript II Reverse Transcriptase (In-
vitrogen). Primer sequences are available in Table S3
using a SYBR GREEN PCR Master Mix (Applied Biosystems,
Foster City, CA) and the following program: 95 °C for 2 min,
40 cycles of 95 °C for 15 s, and 60 °C for 1 min. PCR products
were quantified in real time using an ABI PRISM 7900HT
unexposed D. magna was used to create standard curves for
each primer set. PCR reactions were done in triplicate and
levels were used to normalize for cDNA content.
of the D. magna IRE, a 40-nucleotide RNA oligomer was
designed based on the 5′ region of the D. magna ferritin
mRNA sequence (AJ292556): UCUGUUUUGCUUCGCCAGU-
GUGUGAACAAGCAGUUUCUAC. The 40-nucleotide oligo-
mer was purchased from HHMI/Keck Biotech at Yale
University, deprotected and desalted according to the
manufacturer’s guidelines. The RNA was further purified by
PAGE on a 15%, 8 M urea gel. The 40-nt RNA band was
the gel by incubating the gel fragment in water overnight.
The RNA was concentrated by ethanol precipitation. For
with T4 Kinase (New England Biolabs, Beverly, MA) and
purified by G-50 spin column.
VOL. 41, NO. 3, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY91045
Electromobility Shift Assay. A 0.72-pmol aliquot of RNA
40-mer oligo was denatured at 90 °C for 5 min and allowed
to reanneal by slow cooling to room temperature in 40 mM
HEPES pH 7.2 with 100 mM KCl. The reannealed RNA was
incubated for 30 min with the indicated amount of recom-
University of Chicago, IL) in 4% glycerol, 2% ?-mercapto-
ethanol, and 4 mM MgCl2. The RNA-protein complex was
polyacrylamide gel with 0.5× TBE buffer at 5V/cm for 1.5 h,
analysis (Molecular Dynamics, Sunnyvale, CA). The percent
complex was determined by dividing the band intensity of
the RNA-protein complex by the total radioactivity in the
of Zou and Bonvillian (15). Total protein content was
determined using the BioRad protein assay based on the
Bradford method (16).
Chitinase Assay. To determine if the downregulation of
the exoskeleton genes resulted in a decrease in chitinase
enzyme activity, a chitinase assay was performed. Chitinase
activity was assayed according to the method of Zou and
Bonvillain (15) using 0.2 mg/mL protein extract and the
substrate 4-nitrophenyl N,N′-diacetyl-?-D-chitobioside (Sigma-
Aldrich). Each protein extract was assayed three times and
0.007 unit/mL chitinase from Streptomyces griseus (Sigma-
Aldrich) was used as a positive control.
Results and Discussion
Identification of Gene Expression Profiles and Candidate
gene expression responses to an adverse chronic outcome,
we exposed D. magna to the 1/10 LC50for the microarray
studies (see Table S2). This concentration is below levels
that cause acute lethal toxicity, which may produce a
is greater than the chronic EC50for Cd and Zn and close to
to the 1/10 LC50of Cu, Cd, or Zn, RNA from D. magna was
extracted and hybridized to the 5000-element cDNA mi-
croarray. Differentially expressed clones were sequenced,
and preliminary analysis of these cDNAs reveals that each
metal had a unique expression profile as shown in Figure 1.
A few genes are differentially expressed in response to more
than one metal; however, the majority is specific for each
BLAST searches and PredictProtein, and organized the
predicted proteins by function (Table 1). (Table S4, Sup-
porting Information, provides the complete list of protein
homologues for each differentially expressed cDNA.)
We selected candidate biomarkers of exposure and
confirmed their differential expression using real-time PCR
(RT-PCR) (Table S5, Supporting Information). For many
For others, including ferritin (AJ292556), and the inositol
FIGURE 1. Copper, cadmium, and zinc causing the differential
expression of a unique set of cDNAs. The number of differentially
are shown including the genes wthat overlap in expression by
more than one metal.
TABLE 1. Predicted Function of Differentially Expressed Genes
after Exposure to Copper, Cadmium, or Zinc at the 1/10 LC501
10469ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 3, 2007
expression when the microarray results did not. Further
replication would be needed to confirm the differential
results. The inositol monophosphatase (DV437806) still
remains a promising biomarker of exposure to Cu because
the differential expression in response to the other metals
was in the opposite direction.
of two putative metallothionein (MT) cDNAs, DV437826
and DV437799, which were induced following metal ex-
posure (Figure S1, Supporting Information). MTs are low
molecular weight proteins involved in the transport and
detoxification of heavy metals and have been used as
biomarkers of exposure to metals for many years (17). De-
spite the interest in using D. magna and other daphnids
in ecotoxicology, no MT genes have been cloned from
these organisms. The translated sequences for DV437826
and DV437799 identified in this study create short pep-
tides with an abundance of cysteine residues, many of them
found in the characteristic C-X-C motif of MT proteins,
although it appears we only identified a partial sequence for
DV437799. They share other properties with MTs including
no aromatic residues, no histidine residues, and a low
molecular weight (18) and were also identified as probable
are distinct from other crustacean MTs (Figure S1A). Only a
between the D. magna and other crustacean MTs and the
D. magna MTs do not cluster with other crustacean MTs
Another metal binding protein and potential biomarker
gene, AJ292556 (NCBI accession number). It is similar to the
copper and cadmium exposure (Table 1 and Table S4), and
(Table S5). Numerous ferritins characterized in insects are
involved in the storage and scavenging of iron and are
transcriptionally and translationally upregulated by Fe (20).
Ferritin is also regulated by oxidative stress (21), and ferritin
mRNA induction by Cd was shown in Xenopus leavis (22).
The transcriptional induction of ferritin therefore, may be
an indirect effect, mediated by oxidative stress caused by
Prior to this study, only the partial sequence of the D.
magna ferritin was known. When we investigated the 5′
untranslated region of the EST sequence for this gene, we
found an iron-response element (IRE) (Figure 2). Transla-
tional control of ferritins in many species has been shown
to be the result of iron-response protein (IRP)/IRE interac-
tions. IRPs bind to the hairpin structure created by the IRE,
which rests upstream of the ferritin start codon, and inhibit
translation of the ferritin protein. Other factors including
to affect IRP binding (23). IREs have been characterized in
multiple species including the crustacean, Pacifastacus
in nucleotide sequence between the D. magna IRE, the P.
IREs. Figure 2B shows the predicted secondary structure of
the D. magna IRE, which forms the typical hairpin loop to
which an IRP can bind. To determine if the D. magna IRE
is able to bind IRP1, we performed an electromobility shift
assay with recombinant rabbit IRP1. As shown in Figure 2C,
IRP1 binds to the D. magna IRE and the extent of complex
formation is dependent on the protein concentration,
D. magna cDNAs obtained from the Daphnia genome
database wFleaBase (http://wfleabase.org/), WFes0007968
The functionality of the D. magna IRE and the presence of
IRP1 homologues in D. magna provide strong evidence that
D. magna ferritin is under the translational control of iron
Prediction of Toxic Modes of Action. By inspecting the
expression data for patterns that could predict modes of
action and biological pathways affected by metal exposure,
we identified four possible mechanisms: (1) the effect of
metals on digestion, (2) possible oxidative stress caused by
Cd, (3) immune suppression induced by Cu, and (4) Zn’s
influence on chitinase activity.
The only genes downregulated by all three metals are all
all three metals caused a repression of the R-amylase
homologue by RT-PCR (Table S5). Studies have shown a
slowing of digestion and a suppression of feeding rates after
exposure to Cd or Zn (25, 26). These observations were
that the observed decrease in enzymatic activity was caused
by a decrease in the expression of the enzymes (25). An
investigation of the chronic effects of metals to D. magna
FIGURE 2. 5′ Untranslated region (UTR) of the D. magna ferritin
mRNA containing a functional IRE. (A) Sequence of the D. magna
IRE is similar to IREs found in the crayfish, P. leniusculus, and the
consensus IRE sequence of insect ferritins (20). (B) The secondary
structure of the D. magna IRE as predicted by Mfold (38). (C) Rabbit
IRP1 gel shift experiment. 5′-32P-labeled D. magna IRE 40-nt 7.8 nM
separated from the complex by 4% (19:1) acrylamide gel electro-
full binding at the RNA-protein ratio of 1:32. The gel shown is a
representation of three experiments with two different protein
has been shown previously (39).
VOL. 41, NO. 3, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY91047
organ including shrinking and paralysis (27). These effects
suggest an overall dysfunction of the D. magna digestive
system that would likely cause a decrease in expression of
Three genes potentially involved in the oxidative stress
response are upregulated by Cd exposure. These genes
include DV437830 and DV437833 (homologues to glu-
tathione-S-transferases, GST) and DV437829 (a homolog to
peroxiredoxin V) (see Tables 1 and S4). We confirmed the
cells from oxidative damage and has been shown to be
increased in D. magna by Cd exposure (29).
cytotoxicity in tissue culture (30). The D. magna gene
DV437829 is similar to peroxiredoxins found in yeast and
insects and contains a conserved cysteine residue found in
the catalytic site of peroxiredoxins (Figure S2).
Two genes possibly involved in immune response,
DV437823 and DV437821, were downregulated by copper
exposure (Table 1), and the downregulation of DV437823
was confirmed by RT-PCR (Table S5). The proteins encoded
by these genes show similarities to ?-1,3-glucan binding
proteins (LGBP) and lectins, proteins involved in the
arthropod innate immune response. They are responsible
for the recognition of an infection and are the first actors in
a cascade of events leading to the activation of the pro-
in invertebrates (31). While infection causes an increased
expression of these proteins (32), one would expect that a
decrease in expression levels would lead to a suppression of
the immune response. Indeed, studies have shown an
impairment of immune factors and a higher susceptibility
to infection in crustaceans and mollusks after exposure to
sublethal concentrations of copper (33, 34). It seems reason-
able that the decrease in expression of ?-1,3-glucan binding
could be responsible for the immune system suppression
seen in copper-exposed organisms. Further confirmation of
the invertebrate immune system.
Two genes (DV437857 and DV437858) downregulated by
zinc code for homologues to chitinase proteins that contain
MDWEYP (35). The only other gene shown to be uniquely
(Table S5), whose translated sequence contains repeats of
AAPA, a motif commonly found in cuticle proteins (36). To
further confirm the influence of zinc on chitin metabolism,
in chitinase activity compared to the control and this effect
was not seen from exposure to Cu or Cd. The decrease in
chitinase activity is dose-dependent with 100 µg/L causing
with chronic effects to reproduction, which are first evident
see a 40% decrease, which appears to be highest level of
suppression before reaching acutely toxic concentrations
above the NOEC of 1000 µg/L.
decrease in enzyme activity suggests that zinc is interfering
with exoskeleton maintenance and molting. Molting is a
highly controlled process in arthropods regulated by hor-
mones including 20-hydroxyecdysone (35). Toxicants in-
cluding PCBs, endosulfan, and DES inhibit molting in D.
Chitinase activity has also been shown to be hormone-
regulated, and certain xenobiotics can inhibit chitinase
activity (15); however, to our knowledge, no studies have
must shed their exoskeleton in order to release a new brood
of neonates, zinc may be interfering with reproduction. We
have shown that the decrease in chitinase activity correlates
with chronic effects on reproduction. Whether these effects
question for future studies.
We have shown that three metals, toxicants of the same
chemical class, have distinct expression profiles in a classic
ecotoxicology test organism, D. magna. These expression
patterns are robust, confirmed by an independent method,
RT-PCR, and correlate well with existing knowledge about
FIGURE 3. Decrease in chitinase activity caused by zinc that is dose dependent. The total chitinase activity was measured following
24-h exposure to Cu, Cd, or Zn, normalized by total protein content, and divided by the chitinase activity in the nonexposed control to
determine the percent activity relative to the control. (A). D. magna were exposed to the 1/10 LC50for each metal: Cu (6 µg/L), Cd (18 µg/L),
and Zn (500 µg/L). (B). A dose response was performed from 100 µg/L Zn to the NOEC of 1000 µg/L Zn.
10489ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 3, 2007
toxicants. The differentially expressed genes identified can
be used as biomarkers of exposure and may be used in field
studies to detect metals in water. Applying genomic tools
such as cDNA microarrays to ecotoxicology will require
further validation and faces obstacles ahead, but this study
has provided evidence for the feasibility of ecotoxicoge-
Special thanks to John Colbourne, Indiana University, for
providing the D. magna cDNA library, William Walden,
University of Chicago, for providing rabbit IRP1 and Henri
was supported by USEPA STAR fellowship (FP-91644201-0),
National Institute of Health (NIH-DK20251), and U.S. Army
Engineer Research and Development Center (BAA053799).
This work benefits from, and contributes to the Daphnia
Supporting Information Available
Toxicity data (Table S1), details on water parameters (Table
S2) and RT-PCR primers (Table S3), a complete list of
differentially expressed genes by each metal (Table S4), and
real-time PCR results (Table S5). Sequence alignments of D.
S2). This material is available free of charge via the Internet
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Received for review June 30, 2006. Revised manuscript re-
ceived October 13, 2006. Accepted October 30, 2006.
10509ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 3, 2007