William S. Branham

QSAR Models Using a Large Diverse Set of Estrogens

Endocrine disruptors (EDs) have a variety of adverse effects in humans and animals. About 58,000 chemicals, most having little safety data, must be tested in a group of tiered assays. As assays will take years, it is important to develop rapid methods to help in priority setting. For application to large data sets, we have developed an integrated system that contains sequential four phases to predict the ability of chemicals to bind to the estrogen receptor (ER), a prevalent mechanism for estrogenic EDs. Here we report the results of evaluating two types of QSAR models for inclusion in phase III to quantitatively predict chemical binding to the ER. Our data set for the relative binding affinities (RBAs) to the ER consists of 130 chemicals covering a wide range of structural diversity and a 6 orders of magnitude spread of RBAs. CoMFA and HQSAR models were constructed and compared for performance. The CoMFA model had a r2 = 0.91 and a q2LOO = 0.66. HQSAR showed reduced performance compared to CoMFA with r2 = 0.76 and q2LOO = 0.59. A number of parameters were examined to improve the CoMFA model. Of these, a phenol indicator increased the q2LOO to 0.71. When up to 50% of the chemicals were left out in the leave-N-out cross-validation, the q2 remained significant. Finally, the models were tested by using two test sets; the q2pred for these were 0.71 and 0.62, a significant result which demonstrates the utility of the CoMFA model for predicting the RBAs of chemicals not included in the training set. If used in conjunction with phases I and II, which reduced the size of the data set dramatically by eliminating most inactive chemicals, the current CoMFA model (phase III) can be used to predict the RBA of chemicals with sufficient accuracy and to provide quantitative information for priority setting.

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.

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.

Microarray scanner calibration curves: characteristics and implications

BackgroundMicroarray-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.ResultsBy 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.ConclusionIt 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.

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.

Differential Sensitivity of Rat Uterine Growth and Epithelium Hypertrophy to Estrogens and Antiestrogens

Abstract Triphenylethylene antiestrogens are considered weak estrogen agonists based on their limited ability to induce estrogen responses, in particular uterine growth. We compared the uterotrophic activity of naturally occurring and synthetic estrogens with that of antiestrogens by quantitating uterine wet weight and hypertrophy in the uterine luminal and glandular epithelium. Immature rats received five daily injections of either an estrogen (17β-estradiol [E2], diethylstilbestrol [DES], or ethynyl estradiol [EE]) or an antiestrogen (tamoxifen [TAM], monohydroxytamoxifen [OH-TAM], or clom-iphene citrate [CC]) (0.001-100 μg/rat/day) subcutaneously in sesame oil and were sacrificed approximately 2 hr after the last injection. Both DES and EE increased uterine weight at doses between 0.01-100 μg/rat/day; E2 was about 10-fold less potent. The antiestrogens increased uterine weight only slightly. DES, EE, and the three antiestrogens each increased luminal epithelium hypertrophy to over 3-fold above that in controls. While the potencies of these synthetic compounds differed (DES = EE > OH-TAM > TAM = CC), each hypertrophic response occurred over two log doses, and the response curves displayed identical slopes. E2, however, required a range of four log doses to achieve the same degree of luminal epithelium hypertrophy. The three antiestrogens elicited glandular epithelium hypertrophy up to 2-fold above controls at the same doses that induced luminal epithelium hypertrophy; the order of potency was OH-TAM > TAM = CC. However, the three estrogens increased glandular epithelium hypertrophy only marginally. Thus, under dosing conditions commonly used to assess uterotrophic activity, these “antiestrogens” are complete, albeit less potent, estrogen agonists in the luminal epithelium and, unlike estrogens, induce hypertrophy in the glandular epithelium.

Transgenic expression of proximal tubule peroxisome proliferator–activated receptor-α 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.

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

BackgroundEnvironmental 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.ResultsOzone in central Arkansas typically ranges between ~22 ppb to ~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 ~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.ConclusionSimple 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.

The Effects of Phytoestrogens on Neonatal Rat Uterine Growth and Development

Abstract Phytoestrogens found in clover, alfalfa, and soybeans have caused reproductive toxicity in several mammalian species. Other estrogens, such as diethylstilbestrol (DES), are developmental toxicants, reducing uterine estrogen receptor (ER) concentration, altering uterine growth, and eliciting reproductive tract abnormalities in the rat. The present study examines the effects of the phytoestrogens coumestrol and equol on the developing rat uterus. Various doses of these compounds were injected sc on postnatal days (PND) 1-5 or 1-10 to ascertain their effects on uterine weight and ER levels, and on PND 10-14 to determine their effects on uterine weight and gland genesis. Coumestrol (PND 1-5) was about 10-3 as potent as DES in increasing uterine weight (wet or dry) while equol increased dry weight only, with a potency of 10-5 that of DES. Although the 10 and 100 μg doses of coumestrol (PND 1-5 or 1-10) initially increased uterine wet weight, by PND 20 uterine weights either equaled or fell significantly below controls. The 100-μg dose of coumestrol (PND 1-5 or 1-10) reduced ER levels at all ages, while the 10-μg dose was not as effective. Equol (PND 1-5 or 1-10) did not affect ER levels. Premature uterine gland genesis occurred by PND 9 for the PND 1-5 100-μg coumestrol dose. When given on PND 10-14 (the critical period of gland genesis), 10 μg and 100 μg of coumestrol and 10 μg DES greatly increased uterine weight, while no effect was elicited by equol. Although coumestrol and equol inhibited uterine gland genesis in a dose-dependent manner, neither abolished gland genesis as did 10 μg of DES or tamoxifen. These data demonstrate that coumestrol elicits uterine biochemical and morphological toxicity much like DES. Equol decreased uterine gland number without increasing uterine wet weight or luminal epithelial hypertrophy, which is inconsistent with either an estrogenic or antiestrogenic action in the uterus.

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.