Human sex hormone-binding globulin binding affinities of 125 structurally diverse chemicals and comparison with their binding to androgen receptor, estrogen receptor, and α-fetoprotein.

One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical's availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical's potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered.

Clamping instability and van der Waals forces in carbon nanotube mechanical resonators.

We investigate the role of weak clamping forces, typically assumed to be infinite, in carbon nanotube mechanical resonators. Due to these forces, we observe a hysteretic clamping and unclamping of the nanotube device that results in a discrete drop in the mechanical resonance frequency on the order of 5-20 MHz, when the temperature is cycled between 340 and 375 K. This instability in the resonant frequency results from the nanotube unpinning from the electrode/trench sidewall where it is bound weakly by van der Waals forces. Interestingly, this unpinning does not affect the Q-factor of the resonance, since the clamping is still governed by van der Waals forces above and below the unpinning. For a 1 µm device, the drop observed in resonance frequency corresponds to a change in nanotube length of approximately 50-65 nm. On the basis of these findings, we introduce a new model, which includes a finite tension around zero gate voltage due to van der Waals forces and shows better agreement with the experimental data than the perfect clamping model. From the gate dependence of the mechanical resonance frequency, we extract the van der Waals clamping force to be 1.8 pN. The mechanical resonance frequency exhibits a striking temperature dependence below 200 K attributed to a temperature-dependent slack arising from the competition between the van der Waals force and the thermal fluctuations in the suspended nanotube.

A rat RNA-Seq transcriptomic BodyMap across 11 organs and 4 developmental stages.

The rat has been used extensively as a model for evaluating chemical toxicities and for understanding drug mechanisms. However, its transcriptome across multiple organs, or developmental stages, has not yet been reported. Here we show, as part of the SEQC consortium efforts, a comprehensive rat transcriptomic BodyMap created by performing RNA-Seq on 320 samples from 11 organs of both sexes of juvenile, adolescent, adult and aged Fischer 344 rats. We catalogue the expression profiles of 40,064 genes, 65,167 transcripts, 31,909 alternatively spliced transcript variants and 2,367 non-coding genes/non-coding RNAs (ncRNAs) annotated in AceView. We find that organ-enriched, differentially expressed genes reflect the known organ-specific biological activities. A large number of transcripts show organ-specific, age-dependent or sex-specific differential expression patterns. We create a web-based, open-access rat BodyMap database of expression profiles with crosslinks to other widely used databases, anticipating that it will serve as a primary resource for biomedical research using the rat model.

Development of doxorubicin-induced chronic cardiotoxicity in the B6C3F1 mouse model.

Serum levels of cardiac troponins serve as biomarkers of myocardial injury. However, troponins are released into the serum only after damage to cardiac tissue has occurred. Here, we report development of a mouse model of doxorubicin (DOX)-induced chronic cardiotoxicity to aid in the identification of predictive biomarkers of early events of cardiac tissue injury. Male B6C3F(1) mice were administered intravenous DOX at 3mg/kg body weight, or an equivalent volume of saline, once a week for 4, 6, 8, 10, 12, and 14weeks, resulting in cumulative DOX doses of 12, 18, 24, 30, 36, and 42mg/kg, respectively. Mice were sacrificed a week following the last dose. A significant reduction in body weight gain was observed in mice following exposure to a weekly DOX dose for 1week and longer compared to saline-treated controls. DOX treatment also resulted in declines in red blood cell count, hemoglobin level, and hematocrit compared to saline-treated controls after the 2nd weekly dose until the 8th and 9th doses, followed by a modest recovery. All DOX-treated mice had significant elevations in cardiac troponin T concentrations in plasma compared to saline-treated controls, indicating cardiac tissue injury. Also, a dose-related increase in the severity of cardiac lesions was seen in mice exposed to 24mg/kg DOX and higher cumulative doses. Mice treated with cumulative DOX doses of 30mg/kg and higher showed a significant decline in heart rate, suggesting drug-induced cardiac dysfunction. Altogether, these findings demonstrate the development of DOX-induced chronic cardiotoxicity in B6C3F(1) mice.

Rat α-Fetoprotein binding affinities of a large set of structurally diverse chemicals elucidated the relationships between structures and binding affinities.

Endocrine disrupting chemicals interfere with the endocrine system in animals, including humans, to exert adverse effects. One of the mechanisms of endocrine disruption is through the binding of receptors such as the estrogen receptor (ER) in target cells. The concentration of any chemical in serum is important for its entry into the target cells to bind the receptors. α-Fetoprotein (AFP) is a major transport protein in rodent serum that can bind with estrogens and thus change a chemical's availability for entrance into the target cell. Sequestration of an estrogen in the serum can alter the chemical's potential for disrupting estrogen receptor-mediated responses. To better understand endocrine disruption, we developed a competitive binding assay using rat amniotic fluid, which contains very high levels of AFP, and measured the binding to the rat AFP for 125 structurally diverse chemicals, most of which are known to bind ER. Fifty-three chemicals were able to bind the rat AFP in the assay, while 72 chemicals were determined to be nonbinders. Observations from closely examining the relationship between the binding data and structures of the tested chemicals are rationally explained in a manner consistent with proposed binding regions of rat AFP in the literature. The data reported here represent the largest data set of structurally diverse chemicals tested for rat AFP binding. The data assist in elucidating binding interactions and mechanisms between chemicals and rat AFP and, in turn, assist in the evaluation of the endocrine disrupting potential of chemicals.

Evaluation of Hepatic Mitochondria and Hematological Parameters in Zidovudine-Treated B6C3F(1) Mice.

The effects of 12-week exposure to zidovudine (AZT) at 400, 500, and 600 mg/kg/d were examined on expression of 542 mitochondria-related genes and mitochondrial DNA (mtDNA) copy number in the liver of male and female B6C3F(1) mice to understand mitochondrial role in sex-related differences in development of lactic acidosis. Plasma lactate levels and hematologic parameters were also examined. Results indicated increased red blood cell (RBC) count in vehicle-treated controls, whereas a dose-related decline in the RBC count was noted in AZT-treated mice compared to the basal levels before treatments began. These decreases were associated with significant dose-related increases in mean corpuscular volume and mean corpuscular hemoglobin levels. This effect was greater in AZT-treated females compared to males. In both sexes, 12-week AZT or vehicle exposure significantly reduced plasma lactate levels compared to the basal levels. Results also showed modest, but significant, changes in the expression of genes associated with apoptosis and lipid metabolism at 600 mg/kg/d AZT. Neither drug nor sex influenced hepatic mtDNA copy number. Altogether, 12-week AZT exposure as high as 600 mg/kg/d did not impair hepatic mitochondria or induce lactic acidosis in B6C3F(1) mice. However, AZT-mediated hematologic toxicity appeared to be greater in females compared to males.

Expression analysis of hepatic mitochondria-related genes in mice exposed to acrylamide and glycidamide.

Acrylamide (AA) is an industrial chemical that has been extensively investigated for central nervous system (CNS), reproductive, and genetic toxicity. However, AA effects on the liver, a major organ of drug metabolism, have not been adequately explored. In addition, the role of mitochondria in AA-mediated toxicity is still unclear. Changes in expression levels of genes associated with hepatic mitochondrial function of male transgenic Big Blue (BB) mice administered 500 mg/L AA or an equimolar concentration (600 mg/L) of its reactive metabolite glycidamide (GA) in drinking water for 3 and 4 wk, respectively, were examined. Transcriptional profiling of 542 mitochondria-related genes indicated a significant downregulation of genes associated with the 3-beta-hydroxysteroid dehydrogenase family in AA- and GA-treated mice, suggesting a possible role of both chemicals in altering hepatic steroid metabolism in BB mice. In addition, genes associated with lipid metabolism were altered by both treatments. Interestingly, only the parental compound (AA) significantly induced expression levels of genes associated with oxidative phosphorylation, in particular ATP synthase, which correlated with elevated ATP levels, indicating an increased energy demand in liver during AA exposure. Acrylamide-treated mice also showed significantly higher activity of glutathione S-transferase in association with decreased levels of reduced glutathione (GSH), which may imply an enhanced rate of conjugation of AA with GSH in liver. These results suggest different hepatic mechanisms of action of AA and GA and provide important insights into the involvement of mitochondria during their exposures.

Microarray analysis of virulence gene profiles in Salmonella serovars from food/food animal environment.

INTRODUCTION: Rapid, accurate and inexpensive analysis of the disease-causing potential of foodborne pathogens is an important consideration in food safety and biodefense, particularly in developing countries. The objective of this study is to demonstrate the use of a robust and inexpensive microarray platform to assay the virulence gene profiles in Salmonella from food and/or the food animal environment, and then use ArrayTrack™ for data analysis. METHODOLOGY: The spotted array consisted of 69 selected Salmonella-specific virulence gene probes (65bp each). These probes were printed on poly-L-lysine-coated slides. Genomic DNA was digested with Sau3AI, labeled with Cy3 dye, hybridized to the gene probes, and the images were captured and analyzed by GenePix 4000B and ArrayTrack™, a free software developed by Food and Drug Administration (FDA) researchers. RESULTS: Nearly 58% of the virulence-associated genes tested were present in all Salmonella strains tested. In general, genes belonging to inv, pip, prg, sic, sip, spa or ttr families were detected in more than 90% of the isolates, while the iacP, avrA, invH, rhuM, sirA, sopB, sopE or sugR genes were detected in 40 to 80% of the isolates. The gene variability was independent of the Salmonella serotype. CONCLUSIONS: This hybridization array presents an accurate and cost-effective method for evaluating the disease-causing potential of Salmonella in outbreak investigations by targeting a selective set of Salmonella-associated virulence genes.

Similarities and differences in the expression of drug-metabolizing enzymes between human hepatic cell lines and primary human hepatocytes.

In addition to primary human hepatocytes, hepatoma cell lines, and transfected nonhepatoma, hepatic cell lines have been used for pharmacological and toxicological studies. However, a systematic evaluation and a general report of the gene expression spectra of drug-metabolizing enzymes and transporters (DMETs) in these in vitro systems are not currently available. To fill this information gap and to provide references for future studies, we systematically characterized the basal gene expression profiles of 251 drug-metabolizing enzymes in untreated primary human hepatocytes from six donors, four commonly used hepatoma cell lines (HepG2, Huh7, SK-Hep-1, and Hep3B), and one transfected human liver epithelial cell line. A large variation in DMET expression spectra was observed between hepatic cell lines and primary hepatocytes, with the complete absence or much lower abundance of certain DMETs in hepatic cell lines. Furthermore, the basal DMET expression spectra of five hepatic cell lines are summarized, providing references for researchers to choose carefully appropriate in vitro models for their studies of drug metabolism and toxicity, especially for studies with drugs in which toxicities are mediated through the formation of reactive metabolites.

Age and sex dependent changes in liver gene expression during the life cycle of the rat.

BACKGROUND: Age- and sex-related susceptibility to adverse drug reactions and disease is a key concern in understanding drug safety and disease progression. We hypothesize that the underlying suite of hepatic genes expressed at various life cycle stages will impact susceptibility to adverse drug reactions. Understanding the basal liver gene expression patterns is a necessary first step in addressing this hypothesis and will inform our assessments of adverse drug reactions as the liver plays a central role in drug metabolism and biotransformation. Untreated male and female F344 rats were sacrificed at 2, 5, 6, 8, 15, 21, 52, 78, and 104 weeks of age. Liver tissues were collected for histology and gene expression analysis. Whole-genome rat microarrays were used to query global expression profiles. RESULTS: An initial list of differentially expressed genes was selected using criteria based upon p-value (p < 0.05) and fold-change (+/- 1.5). Three dimensional principal component analyses revealed differences between males and females beginning at 2 weeks with more divergent profiles beginning at 5 weeks. The greatest sex-differences were observed between 8 and 52 weeks before converging again at 104 weeks. K-means clustering identified groups of genes that displayed age-related patterns of expression. Various adult aging-related clusters represented gene pathways related to xenobiotic metabolism, DNA damage repair, and oxidative stress. CONCLUSIONS: These results suggest an underlying role for genes in specific clusters in potentiating age- and sex-related differences in susceptibility to adverse health effects. Furthermore, such a comprehensive picture of life cycle changes in gene expression deepens our understanding and informs the utility of liver gene expression biomarkers.

Genomic analysis of microRNA time-course expression in liver of mice treated with genotoxic carcinogen N-ethyl-N-nitrosourea.

BACKGROUND: Dysregulated expression of microRNAs (miRNAs) has been previously observed in human cancer tissues and shown promise in defining tumor status. However, there is little information as to if or when expression changes of miRNAs occur in normal tissues after carcinogen exposure. RESULTS: To explore the possible time-course changes of miRNA expression induced by a carcinogen, we treated mice with one dose of 120 mg/kg N-ethyl-N-nitrosourea (ENU), a model genotoxic carcinogen, and vehicle control. The miRNA expression profiles were assessed in the mouse livers in a time-course design. miRNAs were isolated from the livers at days 1, 3, 7, 15, 30 and 120 after the treatment and their expression was determined using a miRNA PCR Array. Principal component analysis of the miRNA expression profiles showed that miRNA expression at post-treatment days (PTDs) 7 and 15 were different from those at the other time points and the control. The number of differentially expressed miRNAs (DEMs) changed over time (3, 5, 14, 32, 5 and 5 at PTDs 1, 3, 7, 15, 30 and 120, respectively). The magnitude of the expression change varied with time with the highest changes at PTDs 7 or 15 for most of the DEMs. In silico functional analysis of the DEMs at PTDs 7 and 15 indicated that the major functions of these ENU-induced DEMs were associated with DNA damage, DNA repair, apoptosis and other processes related to carcinogenesis. CONCLUSION: Our results showed that many miRNAs changed their expression to respond the exposure of the genotoxic carcinogen ENU and the number and magnitude of the changes were highest at PTDs 7 to 15. Thus, one to two weeks after the exposure is the best time for miRNA expression sampling.

Transgenic expression of proximal tubule peroxisome proliferator-activated receptor-alpha in mice confers protection during acute kidney injury.

Our previous studies suggest that peroxisome proliferator-activated receptor-alpha (PPARalpha) plays a critical role in regulating fatty acid beta-oxidation in kidney tissue and this directly correlated with preservation of kidney morphology and function during acute kidney injury. To further study this, we generated transgenic mice expressing PPARalpha in the proximal tubule under the control of the promoter of KAP2 (kidney androgen-regulated protein 2). Segment-specific upregulation of PPARalpha expression by testosterone treatment of female transgenic mice improved kidney function during cisplatin or ischemia-reperfusion-induced acute kidney injury. Ischemia-reperfusion injury or treatment with cisplatin in wild-type mice caused inhibition of fatty-acid oxidation, reduction of mitochondrial genes of oxidative phosphorylation, mitochondrial DNA, fatty-acid metabolism, and the tricarboxylic acid cycle. Similar injury in testosterone-treated transgenic mice resulted in amelioration of these effects. Similarly, there were increases in the levels of 4-hydroxy-2-hexenal-derived lipid peroxidation products in wild-type mice, which were also reduced in the transgenic mice. Similarly, necrosis of the S3 segment was reduced in the two injury models in transgenic mice compared to wild type. Our results suggest proximal tubule PPARalpha activity serves as a metabolic sensor. Its increased expression without the use of an exogenous PPARalpha ligand in the transgenic mice is sufficient to protect kidney function and morphology, and to prevent abnormalities in lipid metabolism associated with acute kidney injury.

Effect of (+)-usnic acid on mitochondrial functions as measured by mitochondria-specific oligonucleotide microarray in liver of B6C3F1 mice.

Usnic acid is a lichen metabolite used as a weight-loss dietary supplement due to its uncoupling action on mitochondria. However, its use has been associated with severe liver disorders in some individuals. Animal studies conducted thus far evaluated the effects of usnic acid on mitochondria primarily by measuring the rate of oxygen consumption and/or ATP generation. To obtain further insight into usnic acid-mediated effects on mitochondria, we examined the expression levels of 542 genes associated with mitochondrial structure and functions in liver of B6C3F(1) female mice using a mitochondria-specific microarray. Beginning at 8 weeks of age, mice received usnic acid at 0, 60, 180, and 600 ppm in ground, irradiated 5LG6 diet for 14 days. Microarray analysis showed a significant effect of usnic acid on the expression of several genes only at the highest dose of 600 ppm. A prominent finding of the study was a significant induction of genes associated with complexes I through IV of the electron transport chain. Moreover, several genes involved in fatty acid oxidation, the Krebs cycle, apoptosis, and membrane transporters were over-expressed. Usnic acid is a lipophilic weak acid that can diffuse through mitochondrial membranes and cause a proton leak (uncoupling). The up-regulation of complexes I-IV may be a compensatory mechanism to maintain the proton gradient across the mitochondrial inner membrane. In addition, induction of fatty acid oxidation and the Krebs cycle may be an adaptive response to uncoupling of mitochondria.

Underlying mitochondrial dysfunction triggers flutamide-induced oxidative liver injury in a mouse model of idiosyncratic drug toxicity.

Flutamide, a widely used nonsteroidal anti-androgen, but not its bioisostere bicalutamide, has been associated with idiosyncratic drug-induced liver injury. Although the susceptibility factors are unknown, mitochondrial injury has emerged as a putative hazard of flutamide. To explore the role of mitochondrial sensitization in flutamide hepatotoxicity, we determined the effects of superimposed drug stress in a murine model of underlying mitochondrial abnormalities. Male wild-type or heterozygous Sod2(+/-) mice were injected intraperitoneously with flutamide (0, 30 or 100 mg/kg/day) for 28 days. A kinetic pilot study revealed that flutamide (100 mg/kg/day) caused approximately 10-fold greater exposure than the reported therapeutic mean plasma levels. Mutant (5/10), but not wild-type, mice in the high-dose group exhibited small foci of hepatocellular necrosis and an increased number of apoptotic hepatocytes. Hepatic GSSG/GSH, protein carbonyl levels, and serum lactate levels were significantly increased, suggesting oxidant stress and mitochondrial dysfunction. Measurement of mitochondrial superoxide in cultured hepatocytes demonstrated that mitochondria were a significant source of flutamide-enhanced oxidant stress. Indeed, mitochondria isolated from flutamide-treated Sod2(+/-) mice exhibited decreased aconitase activity as compared to vehicle controls. A transcriptomics analysis using MitoChips revealed that flutamide-treated Sod2(+/-) mice exhibited a selective decrease in the expression of all complexes I and III subunits encoded by mitochondrial DNA. In contrast, Sod2(+/-) mice receiving bicalutamide (50 mg/kg/day) did not reveal any hepatic changes. These results are compatible with our concept that flutamide targets hepatic mitochondria and exerts oxidant stress that can lead to overt hepatic injury in the presence of an underlying mitochondrial abnormality.

Effect of short-term exposure to zidovudine (AZT) on the expression of mitochondria-related genes in skeletal muscle of neonatal mice.

Zidovudine (3'-azido-3'-deoxythymidine; AZT) is the main anti-retroviral drug given to HIV-1-infected pregnant women during pregnancy and to their infants after birth to reduce mother-to-child transmission of the virus. In animal studies, however, a significant mitochondrial morphological damage has been reported in skeletal muscle as a consequence of transplacental or perinatal exposure to AZT. Because proper muscle function is highly dependent on efficient mitochondrial function and information on AZT-induced mitochondrial toxicity during neonatal exposure is limited, we investigated the effect of AZT on the expression of 542 mitochondria-related genes encoded by both nuclear and mitochondrial DNA in the skeletal muscle of infant male and female mice using microarray technology. Animals were treated orally by gavage with AZT at 0, 10, 50, 100, and 200mg/kg body weight/day from postnatal day (PND) 1 through 8 and were sacrificed at 1- and 2-h following the last dose on PND 8. These doses in mice correspond to 0, 1.1, 5.5, 11.0, and 22.0mg/kg AZT in human infants [Center for Drug Evaluation and Research (CDER) 2005. Pharmacology and Toxicology, Guidance for industry. Estimating the maximum safe dose in initial clinical trials for therapeutics in adult healthy volunteers, p. 7. http://www.fda.gov/cder/guidance/index.htm.]. Microarray data were analyzed for effects of time, sex, treatment, and their interactions using a fixed effect linear model. The results showed modest, but significant, dose-related responses in the expression level of genes associated with apoptosis, fatty acid metabolism, mitochondrial DNA maintenance, and various mitochondrial membrane transporters. The transcription levels were not significantly different at both time points and were not sex dependent. The results suggest that changes in expression of mitochondria-related genes in skeletal muscle may be an initial response to short-term AZT exposure in infant mice.

Nucleoside reverse transcriptase inhibitors (NRTIs)-induced expression profile of mitochondria-related genes in the mouse liver.

Mitochondrial dysfunction has been implicated in the adverse effects of nucleoside reverse transcriptase inhibitors (NRTIs) used to treat HIV-1 infections. To gain insight into the mechanism by which NRTIs alter mitochondrial function, the expression level of 542 genes associated with mitochondrial structure and functions was determined in the livers of p53 haplodeficient (+/-) C3B6F1 female mouse pups using mouse mitochondria-specific oligonucleotide microarray. The pups were transplacentally exposed to zidovudine (AZT) at 240 mg/kg bw/day or a combination of AZT and lamivudine (3TC) at 160 and 100mg/kg bw/day, respectively, from gestation day 12 through 18, followed by continuous treatment by oral administration from postnatal day 1-28. In addition, AZT/3TC effect was investigated in wild-type (+/+) C3B6F1 female mice. The genotype did not significantly affect the gene expression profile induced by AZT/3TC treatment. However, the transcriptional level of several genes associated with oxidative phosphorylation, mitochondrial tRNAs, fatty acid oxidation, steroid biosynthesis, and a few transport proteins were significantly altered in pups treated with AZT and AZT/3TC compared to their vehicle counterparts. Interestingly, AZT/3TC altered the expression level of 153 genes with false discovery rate of less than 0.05, in contrast to only 20 genes by AZT alone. These results suggest that NRTI-related effect on expression level of genes associated with mitochondrial functions was much greater in response to AZT/3TC combination treatment than AZT alone.

Development of mitochondria-specific mouse oligonucleotide microarray and validation of data by real-time PCR.

This study describes the development of a mitochondria-specific microarray, MitoChip, to measure transcripts of mitochondria-associated genes in various diseases and drug-induced toxicities in the mouse. The array consists of 542 oligonucleotides that represent genes from the mitochondrial and nuclear genomes associated with mitochondrial structure and functions. The expression of mitochondrial genes was measured in the liver of both p53 haplodeficient (+/-) and wild-type (+/+) C3B6F(1) female mice exposed to antiretroviral agents, Zidovudine (AZT) and Lamivudine (3TC). Among genes whose expression was significantly altered, a set was selected for real-time PCR analysis to verify their differential gene expression. The real-time PCR data confirmed the observations by microarray analysis suggesting that the MitoChip may be an important tool for examining mitochondrial involvement in diseases and drug-induced toxicities.

Elimination of laboratory ozone leads to a dramatic improvement in the reproducibility of microarray gene expression measurements.

BACKGROUND: Environmental ozone can rapidly degrade cyanine 5 (Cy5), a fluorescent dye commonly used in microarray gene expression studies. Cyanine 3 (Cy3) is much less affected by atmospheric ozone. Degradation of the Cy5 signal relative to the Cy3 signal in 2-color microarrays will adversely reduce the Cy5/Cy3 ratio resulting in unreliable microarray data. RESULTS: Ozone in central Arkansas typically ranges between approximately 22 ppb to approximately 46 ppb and can be as high as 60-100 ppb depending upon season, meteorological conditions, and time of day. These levels of ozone are common in many areas of the country during the summer. A carbon filter was installed in the laboratory air handling system to reduce ozone levels in the microarray laboratory. In addition, the airflow was balanced to prevent non-filtered air from entering the laboratory. These modifications reduced the ozone within the microarray laboratory to approximately 2-4 ppb. Data presented here document reductions in Cy5 signal on both in-house produced microarrays and commercial microarrays as a result of exposure to unfiltered air. Comparisons of identically hybridized microarrays exposed to either carbon-filtered or unfiltered air demonstrated the protective effect of carbon-filtration on microarray data as indicated by Cy5 and Cy3 intensities. LOWESS normalization of the data was not able to completely overcome the effect of ozone-induced reduction of Cy5 signal. Experiments were also conducted to examine the effects of high humidity on microarray quality. Modest, but significant, increases in Cy5 and Cy3 signal intensities were observed after 2 or 4 hours at 98-99% humidity compared to 42% humidity. CONCLUSION: Simple installation of carbon filters in the laboratory air handling system resulted in low and consistent ozone levels. This allowed the accurate determination of gene expression by microarray using Cy5 and Cy3 fluorescent dyes.

Improvement in the reproducibility and accuracy of DNA microarray quantification by optimizing hybridization conditions.

BACKGROUND: DNA microarrays, which have been increasingly used to monitor mRNA transcripts at a global level, can provide detailed insight into cellular processes involved in response to drugs and toxins. This is leading to new understandings of signaling networks that operate in the cell, and the molecular basis of diseases. Custom printed oligonucleotide arrays have proven to be an effective way to facilitate the applications of DNA microarray technology. A successful microarray experiment, however, involves many steps: well-designed oligonucleotide probes, printing, RNA extraction and labeling, hybridization, and imaging. Optimization is essential to generate reliable microarray data. RESULTS: Hybridization and washing steps are crucial for a successful microarray experiment. By following the hybridization and washing conditions recommended by an oligonucleotide provider, it was found that the expression ratios were compressed greater than expected and data analysis revealed a high degree of non-specific binding. A series of experiments was conducted using rat mixed tissue RNA reference material (MTRRM) and other RNA samples to optimize the hybridization and washing conditions. The optimized hybridization and washing conditions greatly reduced the non-specific binding and improved the accuracy of spot intensity measurements. CONCLUSION: The results from the optimized hybridization and washing conditions greatly improved the reproducibility and accuracy of expression ratios. These experiments also suggested the importance of probe designs using better bioinformatics approaches and the need for common reference RNA samples for platform performance evaluation in order to fulfill the potential of DNA microarray technology.

Microarray scanner calibration curves: characteristics and implications.

BACKGROUND: Microarray-based measurement of mRNA abundance assumes a linear relationship between the fluorescence intensity and the dye concentration. In reality, however, the calibration curve can be nonlinear. RESULTS: By scanning a microarray scanner calibration slide containing known concentrations of fluorescent dyes under 18 PMT gains, we were able to evaluate the differences in calibration characteristics of Cy5 and Cy3. First, the calibration curve for the same dye under the same PMT gain is nonlinear at both the high and low intensity ends. Second, the degree of nonlinearity of the calibration curve depends on the PMT gain. Third, the two PMTs (for Cy5 and Cy3) behave differently even under the same gain. Fourth, the background intensity for the Cy3 channel is higher than that for the Cy5 channel. The impact of such characteristics on the accuracy and reproducibility of measured mRNA abundance and the calculated ratios was demonstrated. Combined with simulation results, we provided explanations to the existence of ratio underestimation, intensity-dependence of ratio bias, and anti-correlation of ratios in dye-swap replicates. We further demonstrated that although Lowess normalization effectively eliminates the intensity-dependence of ratio bias, the systematic deviation from true ratios largely remained. A method of calculating ratios based on concentrations estimated from the calibration curves was proposed for correcting ratio bias. CONCLUSION: It is preferable to scan microarray slides at fixed, optimal gain settings under which the linearity between concentration and intensity is maximized. Although normalization methods improve reproducibility of microarray measurements, they appear less effective in improving accuracy.