Sensitive Molecular Biomarkers Research Articles

Atrazine affects kidney and adrenal hormones (AHs) related genes expressions of rare minnow ( Gobiocypris rarus)

Atrazine, one of the most widely used herbicides, has been proved to interfere with sexual hormones. However few studies have considered the effects of atrazine on adrenal hormones (AH). In this study, rare minnow ( Gobiocypris rarus) was exposed to 0, 3, 10, 33, 100 and 333 μg/l atrazine for 28 days. The histopathology of kidney and gill was examined and the expressions of AHs-related genes including Na + ,K + -ATPase, glucocorticoid receptor ( gr), heat shock protein 70 ( hsp70), and heat shock protein 90 ( hsp90) in kidney and gill were quantitatively determined. Histopathological observation revealed obvious lesions in gill including hyperplasia, necrosis in epithelium region, aneurysm and lamellar fusion at concentrations as low as 10 μg/l. The observed lesions in kidney included extensive expansion in the lumen, degenerative and necrotic changes of the tubular epithelia, shrinkage of the glomerulus as well as increase of the Bowman's space at concentrations as low as 10 μg/l. The expressions of Na + ,K + -ATPase, gr, hsp70 and hsp90 in the kidney of females were significantly decreased at all concentrations. For males, the expressions of hsp90 in the kidney of all treated groups were significantly down-regulated, while gr at all concentrations and hsp70 at 10, 33, 100 μg/l were significantly up-regulated. However in the gill, the expressions of these genes were not significantly different from the control. These results indicated that exposure to atrazine caused impairments of kidney and gill of fish at environmental related concentrations. Histopathological lesions could partly attribute to the changes of the expressions of AHs-related genes in kidney. We concluded also that atrazine is a potential AHs-disruptor and AHs-related genes in kidney of fish could be used as sensitive molecular biomarkers.

Choriogenin mRNA as a sensitive molecular biomarker for estrogenic chemicals in developing brackish medaka ( Oryzias melastigma)

Teleost choriogenins, precursors of the inner layer subunits of the egg envelope, are regarded as sensitive biomarkers for estrogenic pollutants. In this study, two full-length cDNAs, omChgH and omChgL, which encode the choriogenin H and L forms, respectively, were isolated from a brackish medaka, Oryzias melastigma. 17β-Estradiol (E2; 10 μg/L)-dependent expression of omChgH and omChgL was observed starting at embryonic stage 34 and restricted exclusively to the liver. In hatchlings, E2 induction of omChgH was stronger than that of omChgL. Static exposure of adult fish to E2 (0, 1, 10, 100, and 500 ng/L), 17α-ethinylestradiol (EE2; 0, 1, 10, 100 and 500 ng/L), 4-nonylphenol (NP; 0, 1, 10, 100, and 200 μg/L), and bisphenol A (BPA; 0, 1, 10, 100, and 200 μg/L) in artificial seawater for 7 days resulted in dose-dependent induction of both genes in the liver. In the male livers, the sensitivity of omChgH to these estrogenic compounds was higher than that of omChgL; the lowest-observed-effect concentrations (LOECs) of E2, EE2, NP, and BPA on omChgH were 10 ng/L, 10 ng/L, 100 μg/L and 100 μg/L, respectively, and on omChgL were 100 ng/L, 100 ng/L, 100 μg/L, and 200 μg/L, respectively. All these suggest that omChgH can be used as a highly sensitive biomarker for monitoring estrogenic chemicals in the marine environment.

The environmental estrogen, 4-nonylphenol modulates brain estrogen-receptor- and aromatase (CYP19) isoforms gene expression patterns in Atlantic salmon (Salmo salar)

The modulations of reproductive biomarker responses, such as vitellogenin (Vtg) and zona radiata protein (Zr-protein) by xenoestrogens are mediated through the estrogen receptors (ERs). The ERalpha and ERbeta belong to the nuclear receptor (NR) superfamily of ligand activated transcription factors that modulate specific gene expression. By interacting with estrogen and estrogen like molecules (including estrogen mimics), the ERs exert a diverse array of biological effects. In addition to their effect as direct agonist to the ERs, the endocrine-disrupting effects of environmental chemicals are sometimes interpreted as interference with steroidogenesis through the aromatase enzyme (CYP19 or P450arom). The aim of this study was to investigate the effects of nonylphenol on the expression patterns of ER and aromatase (P450arom) isoform genes in the brain as sensitive molecular biomarker of response to endocrine disrupting chemicals. Immature Atlantic salmon were exposed to continuous nominal waterborne nonylphenol at 15 microg/L. Brain samples were collected after 7 days exposure. A quantitative RT-PCR method was used to analyze mRNA expression patterns. Nonylphenol caused a significant induction of P450arom and ER isoform patterns in the brain and represent quantitative gene expression biomarkers of exposure to endocrine disrupting chemicals in the environment.

Differential expression of Cu/Zn superoxide dismutase mRNA during exposures to heavy metals in rockbream ( Oplegnathus fasciatus)

A full-length complementary DNA clone (789 bp) encoding Cu/Zn superoxide dismutase (Cu/Zn SOD) was isolated from the liver cDNA library of a marine fish, rockbream ( Oplegnathus fasciatus). The rockbream SOD cDNA contained a 462 bp open reading frame (ORF) encoding 154 amino acids. Deduced amino acid sequence of rockbream SOD shared relatively high identity with teleost (78–91%) and mammalian (64–70%) orthologues. The SOD transcripts were ubiquitously detected in all the tissues examined including brain, gill, intestine, kidney, liver, muscle, skin and spleen determined by RT-PCR and northern dot blot hybridization. Based on semi-quantitative RT-PCR, real-time PCR and/or scanning densitometry of RNA dot blot hybridization, the transcriptional level of SOD gene was differentially affected by various experimental exposures to heavy metals. Intraperitoneal injections (0, 1, 2, 5 or 10 mg CdCl 2 per kg body weight for 48 h) and immersion treatments (0, 25, 50 or 100 μM of cadmium, copper or zinc for 96 h) revealed that (1) the expression of SOD at mRNA level was generally dependent upon the exposure doses of heavy metals, (2) the stimulation of SOD transcripts was more apparent in liver than kidney and gill, and (3) that cadmium was more potent to induce the expression of SOD than copper and zinc. Immersion exposures using high (25 μM Cd) and low (2 or 5 μM) doses of cadmium for prolonged periods up to 14 (high dose) or 28 (low doses) days also showed that the levels of SOD transcripts were higher with longer durations and higher concentrations. Results from the present study indicate that assessment of transcriptional activity of fish SOD gene at mRNA level might be useful as a sensitive molecular biomarker system to address the oxidative stress caused by heavy metal pollutants in fish.

Dietary exposure to Aroclor 1254 alters gene expression in Xenopus laevis frogs

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that contribute to worldwide health problems. Despite data associating PCBs with adverse health effects, decisions to clean up contaminated sites remain controversial. Cleanup decisions are typically based on risk assessment methods that are not sensitive enough to detect subtle changes in health. We have recently shown that gene expression signatures can serve as sensitive molecular biomarkers of exposure and related health effects. Our initial studies were carried out with developing Xenopus laevis tadpoles that were exposed to the PCB mixture Aroclor 1254 (A1254) for 2 days. A1254 was dissolved in dimethyl sulfoxide and added to the aquarium water for rapid loading of PCBs into the tadpole tissue. These studies showed that increases in the expression of specific genes occurred independent of adverse health effects, and decreases in specific genes correlated with the appearance of observable health effects, including decreased survival and gross morphological and behavioral abnormalities. In this report, we extend our previous work to test the use of gene expression signatures as biomarkers in frogs exposed to PCBs through the diet from early tadpole stages through metamorphosis. This work showed that chronic low-dose exposure to A1254 (24ppm) in food produced tissue levels of 17ppm and increased gene expression of nerve growth factor and proopiomelanocortin independent of adverse health effects. Exposure to higher doses of A1254 (200ppm) produced tissue levels of 80ppm and increased expression of p450 1A1, also, independent of adverse health effects. This work provides further evidence for the use of gene expression changes as biomarkers of exposure to PCBs.

Molecular cloning and characterization of a metal responsive Chironomus tentans alpha-tubulin cDNA

Metal pollution of aquatic ecosystems is a problem of economic and health importance. Sensitive molecular biomarkers of metal exposure are sorely needed. We have isolated a cDNA from the midge Chironomus tentans that is transcribed in all organs and developmental stages. The cDNA encodes a protein, designated Chironomus tentans alpha-tubulin 1 (CTTUB1), which has significant similarities with invertebrate and vertebrate alpha-tubulins. CTTUB1 is abundantly transcribed in embryos and to a lesser extent in adults and larvae. CTTUB1 RNA and protein abundances are increased in larvae exposed to copper or cadmium. The pattern of cellular distribution of CTTUB1 protein in the midgut epithelial cells was radically affected by cadmium. In the midgut cells of unexposed larvae, CTTUB1 was found evenly distributed throughout the cytoplasm, while in cadmium-exposed larvae, CTTUB1 was mostly concentrated along the basolateral plasma membrane. A mechanism for the regulation of alpha-tubulin synthesis by cadmium is proposed. This is the first report on the isolation of a metal responsive gene from a neartic aquatic insect.

Molecular basis for effects of carcinogenic heavy metals on inducible gene expression.

Certain forms of the heavy metals arsenic and chromium are considered human carcinogens, although they are believed to act through very different mechanisms. Chromium(VI) is believed to act as a classic and mutagenic agent, and DNA/chromatin appears to be the principal target for its effects. In contrast, arsenic(III) is considered nongenotoxic, but is able to target specific cellular proteins, principally through sulfhydryl interactions. We had previously shown that various genotoxic chemical carcinogens, including chromium (VI), preferentially altered expression of several inducible genes but had little or no effect on constitutive gene expression. We were therefore interested in whether these carcinogenic heavy metals might target specific but distinct sites within cells, leading to alterations in gene expression that might contribute to the carcinogenic process. Arsenic(III) and chromium(VI) each significantly altered both basal and hormone-inducible expression of a model inducible gene, phosphoenolpyruvate carboxykinase (PEPCK), at nonovertly toxic doses in the chick embryo in vivo and rat hepatoma H411E cells in culture. We have recently developed two parallel cell culture approaches for examining the molecular basis for these effects. First, we are examining the effects of heavy metals on expression and activation of specific transcription factors known to be involved in regulation of susceptible inducible genes, and have recently observed significant but different effects of arsenic(III) and chromium(VI) on nuclear transcription factor binding. Second, we have developed cell lines with stably integrated PEPCK promoter-luciferase reporter gene constructs to examine effects of heavy metals on promoter function, and have also recently seen profound effects induced by both chromium(VI) and arsenic(III) in this system. These model systems should enable us to be able to identify the critical cis (DNA) and trans (protein) cellular targets of heavy metal exposure leading to alterations in expression of specific susceptible genes. It is anticipated that such information will provide valuable insight into the mechanistic basis for these effects as well as provide sensitive molecular biomarkers for evaluating human exposure.