Research
Toxicogenomics for Environmental Health Research
Kun Yan Zhu
Environmental stressors, including pesticides, heavy metals, nutrients, and oxygen depletion, are detrimental to organisms and have now become a growing global concern with respect to environmental health and quality (Seo et al. 2006, Geist et al. 2007). Atrazine is an extensively used herbicide routinely detected in many surface and ground waters in the United States. The inherent effects of atrazine and its ability to modify the toxicity of different insecticides have been commonly observed in aquatic animals (Belden and Lydy 2000, Anderson and Lydy 2001, Jin-Clark et al. 2002, Anderson and Zhu 2004, Rakotondravelo et al. 2006a, b). Atrazine can either synergistically or antagonistically affect the toxicity of certain organophosphate insecticides in the aquatic midge (Chironomus tentans, Fig. 1), an ecologically important bioindicator species (Anderson and Zhu 2004). Biochemical and molecular analyses revealed that increased toxicity of certain organophosphates in binary combination with atrazine was due to increased activation of certain organophosphates by cytochrome P450 monooxygenases in midges (Jin-Clark et al. 2002, Anderson and Zhu 2004, Londono et al. 2004).
To further examine the effect of atrazine on midges, we used a genomics-based technique known as restriction fragment differential display (RFDD)-PCR to compare gene expression profiles between atrazine-treated and untreated midges. We isolated 123 differentially expressed cDNA fragments. Many of these putative genes appear to parallel those responsive to hypoxic stress in midges and other aquatic animals (Penttinen and Holopainen 1995, Wu 2002). We further isolated and sequenced two down-regulated hemoglobin cDNAs from atrazine-treated midges. Northern blot analysis showed that the expression of hemoglobin genes was significantly lower in atrazine-treated midges than that in the control following 96-h exposure (Fig. 2). Conversely, atrazine significantly enhanced oxygen consumption in an atrazine concentration- and exposure time-dependent manner. Our study demonstrates for the first time that atrazine, at 
environmentally relevant concentrations, can elevate respiration, possibly eliciting counteractive measures at the transcriptional level to adapt to oxygen deficiency (Anderson et al. 2008).
We recently sequenced 10,368 expressed sequence tags (ESTs) from a midge cDNA library and retained 10,367 high-quality ESTs with an average length of 896 bp after preprocessing. These ESTs were assembled into 2,912 unique sequences including 2,095 singletons and 817 contigs. Blast search resulted in 2,152 putative transcripts that were functionally annotated and sorted into 10 molecular function categories: binding (37.6%), catalytic (37.5), transporter (12.1), structural molecule (6.7), molecular transducer (1.3), antioxidant (1.1), translation regulator (1.1), enzyme regulator (1.0), transcription regulator (1.0), and motor (0.5) activities. Among these ESTs, relatively large number of the transcripts (89) appears to putatively encode different types of hemoglobin involved in oxygen storage and/or transport, which is consistent with the biology of this insect that uses the hemoglobin for oxygen storage and/or transport. Our EST analysis also revealed at least 29 and 11 transcripts putatively encode cytochrome P450 monooxygenases and glutathione S-transferases, respectively. These enzymes play important roles in the biotransformation of toxic chemicals in various organisms. We plan to use these EST data to develop a DNA microarray for gene expression profiling to examine cellular and molecular responses of midgesto various environmental stressors.
Further characterization of the stress-responsive genes can eventually help us: 1) understand the mechanism of stressor’s effects, and cellular and molecular responses to the stressor; 2) develop stress-specific marker genes for assessing environmental quality; and 3) identify effects at higher levels of biological organization, such as communities and ecosystems.
References
Anderson T.D., Lydy M.J. 2001. Increased toxicity to invertebrates associated with a mixture of atrazine and organophosphate insecticides. Environ. Toxicol. Chem. 21: 1507-1514.
Anderson T.D., Zhu K.Y. 2004. Synergistic and antagonistic effects of atrazine on the toxicity of organophophorodithioate and organophosphorothioate insecticides to Chironomus tentans (Diptera: Chironomidae). Pestic. Biochem. Physiol. 80: 54-64.
Anderson T.D., Jin-Clark Y., Begum K., Starkey S.R., Zhu K.Y. 2008. Gene expression profiling reveals decreased expression of two hemoglobin genes associated with increased consumption of oxygen in Chironomus tentans exposed to atrazine: A possible mechanism for adapting to oxygen deficiency. Aquatic Toxicol. 86: 148-156.
Belden J.B., Lydy M.J. 2000. Impact of atrazine on organophosphate insecticide toxicity. Environ. Toxicol. Chem. 19: 2266-2274.
Geist J., Werner I., Eder K.J., Leutenegger C.M. 2007. Comparisons of tissue-specific transcription of stress response genes with whole animal endpoints of adverse effect in striped bass (Morone saxatilis) following treatment with copper and esfenvalerate. Aquatic Toxicol. 85: 28-39.
Jin-Clark Y., Lydy M.J., Zhu K.Y. 2002. Effects of atrazine on chlorpyrifos toxicity in Chironomustentans (Diptera: Chironomidae). Environ. Toxicol. Chem. 21: 598-603.
Londono D.K., Siegfried B.D., Lydy M.J. 2004. Atrazine induction of a family 4 cytochrome P450 gene in Chironomus tentans (Diptera: Chironomidae). Chemosphere 56, 701-706.
Penttinen O-P., Holopainen I.J. 1995. Physiological energetics of a midge, Chironomus riparius Meigen (Insecta: Diptera): Normoxic heat output over the whole life cycle and response of larva to hypoxia and anoxia. Oecologia 103: 419-424.
Rakotondravelo M.L., Anderson T.D., Charlton R.E., Zhu K.Y. 2006a. Sublethal effects of three pesticides on larval survivorship, growth, and macromolecule production in the aquatic midge, Chironomus tentans (Diptera: Chironomidae). Arch. Environ. Contam. Toxicol. 51: 352-359.
Rakotondravelo M.L., Anderson T.D., Charlton R.E., Zhu K.Y. 2006b. Sublethal effects of three pesticides on activities of selected target and detoxification enzymes in the aquatic midge, Chironomus tentans (Diptera: Chironomidae). Arch. Environ. Contam. Toxicol. 51: 360-366.
Seo J.S., Lee K.-W., Rhee J.-S., Hwang D.-S., Lee Y.-M., Park H.G., Ahn I.-Y., Lee J.-S. 2006. Environmental stressors (salinity, heavy metals, H2O2) modulate expression of glutathione reductase (GR) gene from the intertidal copepod Tigriopus japonicus. Aquatic Toxicol. 80: 281-289.
Wu R.S.S. 2002. Hypoxia: from molecular responses to ecosystem responses. Mar. Pollut. Bull. 45: 35-45.
Recent Relevant Publications
Anderson T.D., Zhu K.Y. 2004. Synergistic and antagonistic effects of atrazine on the toxicity of organophophorodithioate and organophosphorothioate insecticides to Chironomus tentans (Diptera: Chironomidae). Pestic. Biochem. Physiol. 80: 54-64.
Rakotondravelo M.L., Anderson T.D., Charlton R.E., Zhu K.Y. 2006a. Sublethal effects of three pesticides on larval survivorship, growth, and macromolecule production in the aquatic midge, Chironomus tentans (Diptera: Chironomidae). Arch. Environ. Contam. Toxicol. 51: 352-359.
Rakotondravelo M.L., Anderson T.D., Charlton R.E., Zhu K.Y. 2006b. Sublethal effects of three pesticides on activities of selected target and detoxification enzymes in the aquatic midge, Chironomus tentans (Diptera: Chironomidae). Arch. Environ. Contam. Toxicol. 51: 360-366.
Anderson T.D., Jin-Clark Y., Begum K., Starkey S.R., Zhu K.Y. 2008. Gene expression profiling reveals decreased expression of two hemoglobin genes associated with increased consumption of oxygen in Chironomus tentans exposed to atrazine: A possible mechanism for adapting to oxygen deficiency. Aquatic Toxicol. 86: 148-156.
Jin-Clark Y., Anderson T.D., Zhu K.Y. 2008. Effect of alachlor and metolachlor on toxicity of chlorpyrifos and major detoxification enzymes in the aquatic midge, Chironomus tentans (Diptera: Chironomidae). Arch. Environ. Contam. Toxicol. (in press).
