Shift In Dose Response Research Articles

Toxicity mechanisms identification via gene set enrichment analysis of time-series toxicogenomics data: impact of time and concentration.

The advance in high-throughput "toxicogenomics" technologies, which allows for concurrent monitoring of cellular responses globally upon exposure to chemical toxicants, presents promises for next-generation toxicity assessment. It is recognized that cellular responses to toxicants have a highly dynamic nature, and exhibit both temporal complexity and dose-response shifts. Most current gene enrichment or pathway analysis lack the recognition of the inherent correlation within time series data, and may potentially miss important pathways or yield biased and inconsistent results that ignore dynamic patterns and time-sensitivity. In this study, we investigated the application of two score metrics for GSEA (gene set enrichment analysis) to rank the genes that consider the temporal gene expression profile. One applies a novel time series CPCA (common principal components analysis) to generate scores for genes based on their contributions to the common temporal variation among treatments for a given chemical at different concentrations. Another one employs an integrated altered gene expression quantifier-TELI (transcriptional effect level index) that integrates altered gene expression magnitude over the exposure time. By comparing the GSEA results using two different ranking metrics for examining the dynamic responses of reporter cells treated with various dose levels of three model toxicants, mitomycin C, hydrogen peroxide, and lead nitrate, the analysis identified and revealed different toxicity mechanisms of these chemicals that exhibit chemical-specific, as well as time-aware and dose-sensitive nature. The ability, advantages, and disadvantages of varying ranking metrics were discussed. These findings support the notion that toxicity bioassays should account for the cells' complex dynamic responses, thereby implying that both data acquisition and data analysis should look beyond simple traditional end point responses.

PC-FACS

PC-FACS

Reduced sensitivity to the locomotor-stimulant effects of cocaine is associated with increased sensitivity to its discriminative stimulus properties

Outbred Long-Evans rats exhibit wide variation in their locomotor response to cocaine. Here, we investigated the relationship between these individual differences and interoceptive effects of cocaine in low cocaine responder (LCR) and high cocaine responder (HCR) phenotypes. Rats were trained to discriminate cocaine (10.0 mg/kg, intraperitoneally) from saline by repeated pairings of injections with one of two response levers. In subsequent tests for stimulus generalization to other cocaine doses (1.25-15.0 mg/kg), LCRs exhibited partial-to-full generalization at 1.85 and 2.5 mg/kg cocaine, respectively, whereas HCRs did not. When the selective 5-HT reuptake inhibitor fluoxetine (5.0 mg/kg) was coadministered with saline or different cocaine doses, we observed similar upward shifts in dose-response in both phenotypes. In contrast, coadministration of the 5-HT2A/2C agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; 0.3 mg/kg) led to partial substitution of DOI for cocaine and enhancement of the stimulus properties of 1.25 mg/kg cocaine in LCRs only. Finally, a retest of cocaine-induced locomotion after discrimination testing revealed marked behavioral sensitization in LCRs and modest changes in behavior in HCRs. Taken together, these results suggest that initial sensitivity to the locomotor-stimulant effects of cocaine is inversely related to its interoceptive properties and that differences in 5-HT systems may contribute to the phenotypic differences observed.

Analgesic synergy between topical opioids and topical non-steroidal anti-inflammatory drugs in the mouse model of thermal pain

The main aim of the study was to examine analgesic effects of the topical opioids and non-steroidal anti-inflammatory drugs (NSAIDs) in a radiant heat tail-flick nociception model. Also, we have tested whether the addition of lauric acid to propylene glycol improves skin permeation for the opioids and NSAIDs. We found that the addition of lauric acid to propylene glycol dramatically improves the penetration of the drugs, measured by the drug's ED 50. We observed a significant dose response shift to the left for all tested drugs. So, morphine's ED 50 was decreased by 19-fold. The duration of the analgesic activity of morphine dissolved in a combination of propylene glycol and lauric acid was much longer compared with the same dose of the drug dissolved in propylene glycol only. Methadone and hydrocodone also produced analgesic activity in this experimental paradigm. We then assessed potential interactions between opioids, ibuprofen and diclofenac using a fixed, low dose of each. The inclusion of either S-ibuprofen or diclofenac to a fixed, low dose of morphine raised the analgesic response from around 20% to 50% and 80%, respectively. Topical methadone and diclofenac alone produced analgesia in 30% of mice. The combination produced analgesia in 100% of mice (100% versus 60%, P < 0.001) and the analgesic effect was observed for 90 min. Alone, topical methadone and S-ibuprofen produced analgesia in 25% and 30% of mice, respectively. The combination elicited analgesia in 100% of mice (100% versus 55%, P < 0.001) and this analgesic effect lasted for 120 min. Our current findings support the supra-additive interaction of topical mu opioids, S-ibuprofen and diclofenac in a model of moderate to severe pain, radiant heat tail-flick assay.

Attenuation of adenosine receptor‐mediated vasorelaxation by L‐NAME in mouse aorta

In BALB/c mouse thoracic aorta with intact endothelium, adenosine and its analogs produce a dose-response (DR) relaxation with a rank order of potency favoring A2B (NECA>CAD>adenosine>> CGS 21680; Talukder et al. LB4, FASEB J. 13(3), 1999). This was further supported by the fact that the A2B-receptor antagonist, alloxazine shifted DR to right for NECA. However, this study did not investigate the role of endothelium in adenosine-mediated relaxation of mouse aorta. Thus, the present study was conducted to investigate the role of endothelium in adenosine receptor-mediated relaxation. In organ bath studies, in intact endothelium, the EC50s for NECA, CAD and adenosine were found to be 0.05, 1.3 and 3.1 x 10−4 M, respectively. However, in the absence of endothelium, the EC50s for NECA, CAD and adenosine were; 0.098, 1.5 and 0.12 x 10−4 M, respectively indicating a shift in DR to the right. The intactness of endothelium in aortic tissue was tested with acetylcholine. NECA-induced DR relaxation was significantly blocked by L-NAME (10−4 M) in endothelium-intact tissues; whereas no inhibition was found in endothelium-denuded tissues. Moreover, the involvement of NO was confirmed by measuring total nitrites and nitrates (NOx) in endothelium-intact and -denuded aorta. NOx levels with L-NAME (10−4 M) alone and in combination with L-arginine (10−4 M) were 59% and 96%, respectively with respect to control (NECA+PE, p<0.05). These data demonstrate that adenosine-receptor mediated relaxation in mouse aorta is partially dependent on endothelium through adenosine A2B-receptor. (Supported by HL 027339)

Phosphorylation-independent β-Arrestin Translocation and Internalization of Leukotriene B4 Receptors

Leukotriene B4 (LTB4) activates the G-protein-coupled receptor leukotriene B4 receptor 1 (BLT1) to mediate a diverse array of cellular responses in leukocytes including chemotaxis, calcium mobilization, degranulation, and gene expression. To determine the role of phosphorylation in BLT1 regulation, we generated mutants of BLT1 in which all of the serine/threonine residues in the C-tail are converted to alanine or to aspartate/glutamate. These mutants expressed in rat basophilic leukemia RBL-2H3 cells bound LTB4 with similar affinity and activated all of the known functional activities of BLT1, albeit at different levels. The conversion of phosphorylation sites to alanine resulted in enhanced G-protein-mediated activities, whereas conversion to aspartate/glutamate resulted in reduced responses and a right shift in dose response, indicating that receptor phosphorylation is a critical regulator of G-protein-mediated pathways. Surprisingly, translocation of beta-arrestin and receptor internalization was completely independent of BLT1 phosphorylation. Real-time analysis of beta-arrestin translocation and receptor internalization using digital fluorescence video microscopy in cells expressing a red fluorescent protein labeled BLT1 and a green fluorescent protein-tagged beta-arrestin confirmed phosphorylation-independent beta-arrestin translocation and internalization of BLT1. In beta-arrestin-deficient mouse embryo fibroblasts, the BLT1 receptors failed to display endosomal localization upon stimulation. In these cells, co-expression of beta-arrestin-green fluorescent protein with BLT1-red fluorescent protein resulted in co-localization of BLT1 and beta-arrestin upon activation. Thus, receptor phosphorylation-dependent mechanisms regulate G-protein-mediated pathways; however, phosphorylation-independent mechanisms regulate beta-arrestin association and internalization of BLT1.

Decreased blood pressure and vascular smooth muscle tone in mice lacking basolateral Na(+)-K(+)-2Cl(-) cotransporter.

The basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) functions in the maintenance of cellular electrolyte and volume homeostasis. NKCC1-deficient (Nkcc1(-/-)) mice were used to examine its role in cardiac function and in the maintenance of blood pressure and vascular tone. Tail-cuff measurements demonstrated that awake Nkcc1(-/-) mice had significantly lower systolic blood pressure than wild-type (Nkcc1(+/+)) mice (114.5 +/- 2.2 and 131.8 +/- 2.5 mmHg, respectively). Serum aldosterone levels were normal, indicating that extracellular fluid-volume homeostasis was not impaired. Studies using pressure transducers in the femoral artery and left ventricle showed that anesthetized Nkcc1(-/-) mice have decreased mean arterial pressure and left ventricular pressure, whereas myocardial contraction parameters were not significantly different from those of Nkcc1(+/+) mice. When stimulated with phenylephrine, aortic smooth muscle from Nkcc1(+/+) and Nkcc1(-/-) mice exhibited no significant differences in maximum contractility and only moderate dose-response shifts. In phasic portal vein smooth muscle from Nkcc1(-/-) mice, however, a sharp reduction in mechanical force was noted. These results indicate that NKCC1 can be important for the maintenance of normal blood pressure and vascular tone.

Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells.

The cysteinyl leukotrienes (cys-LTs) LTC(4), LTD(4), and LTE(4) are a class of peptide-conjugated lipids formed from arachidonic acid and released during activation of mast cells (MCs). We now report that human cord-blood-derived MCs (hMCs) express the CysLT1 receptor, which responds not only to inflammation-derived cys-LTs, but also to a pyrimidinergic ligand, UDP. hMCs express both CysLT1 protein and transcript, and respond to LTC(4), LTD(4), and UDP with concentration-dependent calcium fluxes, each of which is blocked by a competitive CysLT1 receptor antagonist, MK571. Stably transfected Chinese hamster ovary cells expressing the CysLT1 receptor also exhibit MK571-sensitive calcium flux to all three agonists. Both hMCs and CysLT1 transfectants stimulated with UDP are desensitized to LTC(4), but only partially to LTD(4). Priming of hMCs with IL-4 for 5 days enhances their sensitivity to each agonist, but preferentially lowers their threshold for activation by LTC(4) and UDP (approximately 3 log(10)-fold shifts in dose-response for each agonist) over LTD(4) (1.3 log(10)-fold shift), without altering CysLT1 receptor mRNA or surface protein expression, implying the likely induction of a second receptor with CysLT1-like dual ligand specificity. hMCs thus express the CysLT1 receptor, and possibly a closely related IL-4-inducible receptor, which mediate dual activation responses to cys-LTs and UDP, providing an apparent intersection linking the inflammatory and neurogenic elements of bronchial asthma.

Competitive inhibition of NMDA receptor-mediated currents by extracellular calcium chelators.

Calcium chelators have been widely used in electrophysiological recordings of N-methyl-D-aspartate (NMDA) receptor-mediated currents, as well as in studies of excitotoxicity. Intracellularly applied calcium chelators are known to inhibit, at least in part, such calcium-dependent processes as calmodulin-dependent inactivation, calcineurin-dependent desensitization, and rundown of NMDA receptors. On the other hand, the functional consequences and potential nonspecific effects of extracellularly applied chelators have not been extensively investigated. In whole-cell patch-clamp recordings from human embryonic kidney (HEK) 293 cells transiently transfected with recombinant NMDA receptors, we found that addition of calcium chelators such as EGTA shifted the glutamate dose-response curve to the right, from an EC(50) for NR1A/NR2A of 8 microM in 1.8 mM Ca(2+) to approximately 24 microM in a solution containing nominal 0 Ca(2+)/5 mM EGTA and further to approximately 80 microM in 20 mM EGTA. A similar shift in glutamate dose-response was observed for NR1A/NR2B currents. This dose-response shift was not due to a decrease in extracellular Ca(2+) concentration because there was no change in the glutamate EC(50) at Ca(2+) concentrations ranging from 10 mM to nominal 0/200 microM EGTA. Moreover, addition of 5 mM EGTA fully chelated with 6.8 mM Ca(2+) did not produce any shift in the glutamate dose-response curve. We propose that calcium chelators, containing four free carboxyl moieties, competitively inhibit glutamate binding to NMDA receptors.

Temperature dependence of estrogen binding: importance of a subzone in the ligand binding domain of a novel piscine estrogen receptor

The full length estrogen receptor from Oreochromis aureus (OaER) was cloned and expressed in vitro and in vivo as a functional transcription factor. Amino acid residues involved in the thermal stability of the receptor are located at/near subzones β1 and β3, which are highly conserved in other non-piscine species but not in OaER. Hormone binding studies, however, indicate that OaER is thermally stable but exhibited a ∼3-fold reduced affinity for estrogen at elevated temperatures. Transfection of OaER into various cell lines cultured at different temperatures displayed a significant estrogen dose-response shift compared with that of chicken ER (cER). At 37°C, OaER requires ∼80-fold more estrogen to achieve half-maximal stimulation of CAT. Lowering of the incubation temperature from 37°C to 25°C or 20°C resulted in a 4-fold increase in its affinity for estrogen. The thermally deficient transactivation of OaER at temperatures above 25°C was fully prevented by high levels of estrogen. Thus, compared to cER, the OaER exhibits reduced affinity for estrogen at elevated temperature as reflected in its deficient transactivation capability. Amino acid replacements of OaER β3 subzones with corresponding amino acids from cER could partially rescue this temperature sensitivity. The three-dimensional structure of the OaER ligand binding domain (LBD) was modelled based on conformational similarity and sequence homology with human RXRα apo, RARγ holo and ERα LBDs. Unliganded and 17β-estradiol-liganded OaER LBD retained the overall folding pattern of the nuclear receptor LBDs. The residues at/near the subzone β3 of the LBD constitute the central core of OaER structure. Thus, amino acid alteration at this region potentially alters the structure and consequently its temperature-dependent ligand binding properties.

Visceral analgesic tolerance to intrathecal butorphanol in rats.

Recent experimental data suggest that intrathecal (it) kappa-opioid agonists produce profound visceral analgesia. This study investigated the development of visceral analgesic tolerance to it butorphanol, a potent kappa-agonist that has fewer side effects than commonly used it opioids. Understanding of this tolerance could make it butorphanol more effective in treating chronic visceral pain. This was a randomized, controlled animal study involving 80 Sprague-Dawley rats. Rats implanted with lumbar it catheters were infused either with it saline or butorphanol (52 nmol.hr-1) for 96 hr. Six hours afterwards, each rat was challenged once with one of the differing it butorphanol doses to construct dose-response curves. Visceral analgesia was evaluated by the abdominal writhing responses to the acetic acid injected intraperitoneally. The time of the first writhe and the total number of writhes were recorded. For both the saline- and butorphanol-infused groups, a higher challenge dose of it butorphanol produced longer time for the first writhe to occur (P < 0.01, one-way ANOVA), and fewer writhes occurring within 30 min (P < 0.01, one-way ANOVA). However, the dose response curves of the butorphanol-infused groups were shifted rightward (P < 0.001, partial F test). The challenge doses of it butorphanol produced dose-dependent visceral analgesia in both the saline- and butorphanol-infused groups, confirming its efficacy. The butorphanol-infused groups showed dose-response shifts, demonstrating the development of tolerance to this visceral analgesia.

Age-related changes in septal serotonergic, GABAergic and glutamatergic facilitation of retention in SAMP8 mice

SAMP8/TaJf(P8) mouse strain has an inherited age-related impairment of learning and memory with its onset relatively early in its lifespan. Previously, it was reported that cholinergic and glutamatergic drugs injected into the hippocampus after behavioral training showed considerable shifts in the dose that improved retention in mice at 12 compared to 4 months of age. Cholinergic neurons in the septum supply most of the acetylcholine released in the hippocampus. In the present study, we determined if altered functional status of neurotransmission in the septum might account for the decrease in cholinergic and glutamatergic activity in the hippocampus of older SAMP8 mice. After training on footshock avoidance, P8 mice received a drug injection into the septum. Retention was tested 1 week later. The results indicate that bicuculline, GABA-A, and saclofen, GABA-B, receptor antagonist had to be injected at a higher dose in 12- than in 4-month-old mice to improve retention. The serotonergic antagonists, ketanserin and methiothepin, both showed dose–response shifts such that less drug was needed to improve retention in 12- as compared to 4-month-old mice. It required four times more l-glutamate to improve retention in 12- than in 4-month-old mice. Agonists for acetylcholine, dopamine and norepinephrine receptors or an opiate antagonist required little or no change in the dose needed to improve retention in older P8 mice. SAMP8 mice may show an age-related impairment of septohippocampal functioning.

Characterization of a temperature-sensitive mutation in the hormone binding domain of the human estrogen receptor. Studies in cell extracts and intact cells and their implications for hormone-dependent transcriptional activation.

A previous report from this laboratory (Reese, J.C., and Katzenellenbogen, B. S. (1991) J. Biol. Chem. 266, 10880-10887) identified an estrogen receptor (ER) mutant which had a similar binding affinity for estradiol as wild-type ER but displayed a dose-response shift for estradiol in transactivation studies. In this study, we have utilized hormone binding, DNA binding, and gene transfer experiments to further characterize this mutant, which contains an alanine substitution for a cysteine at amino acid 447 in the hormone binding domain of the receptor. Hormone binding studies indicate that the C447A receptor is a temperature-sensitive mutant, whose instability is only apparent at elevated temperatures, and that ligand can stabilize the mutant receptor. Western blot analysis reveals that the temperature-sensitive loss of hormone binding is not attributable to a degradation of receptor protein, but rather is an inactivation of the receptor's hormone binding ability. In addition to the loss in the hormone binding capacity of the C447A mutant, this mutant shows a temperature-sensitive loss in the DNA binding ability of the receptor. Transactivation profiles of the mutant and wild-type receptors demonstrate that incubation of transfected cells with increasing concentrations of estradiol at more ambient temperatures shifts the mutant receptor's dose-response curves to the left, converging on the wild-type curve. Hence, these transactivation studies reveal that the dose-response shift observed for this mutant in cells reflects the measured instability of the hormone binding and DNA interaction of the C447A mutant that can be demonstrated in vitro. In addition, this temperature-sensitive ER mutant is of interest in that its DNA binding is now ligand-dependent with the result that transcriptional activation now parallels receptor occupancy by ligand, which is similar to other steroid hormone receptors.

Halothane Anaesthesia Does Not Modify the Cardiovascular Response to Phenylephrine in Man

Conflicting results exist regarding the ability of halothane to alter the vascular response to alphat adrenergic agonists in animals. Because data from humans are lacking, we studied the haemodynamic response to phenylephrine (PHE) in eight patients about to undergo coronary artery bypass surgery before and during halothane anaesthesia. After obtaining baseline measurements in patients while awake, the responses to PHE infusion at 30, 40, and 50 μg · min−1 were determined. New baseline measurements were made following stabilisation during anaesthesia with halothane, one per cent inspired in oxygen, prior to surgical incision. Then the responses to identical PHE doses were measured again. Halothane did not influence the cardiovascular response to PHE: there was no doseresponse shift for any cardiovascular variable. No arrhythmias or signs of ischaemia were observed. We conclude that one per cent halothane anaesthesia does not attenuate PHE-induced vasoconstriction in man.

Halothane anaesthesia does not modify the cardiovascular response to phenylephrine in man

Conflicting results exist regarding the ability of halothane to alter the vascular response to alpha 1 adrenergic agonists in animals. Because data from humans are lacking, we studied the haemodynamic response to phenylephrine (PHE) in eight patients about to undergo coronary artery bypass surgery before and during halothane anaesthesia. After obtaining baseline measurements in patients while awake, the responses to PHE infusion at 30, 40, and 50 micrograms.min-1 were determined. New baseline measurements were made following stabilisation during anaesthesia with halothane, one per cent inspired in oxygen, prior to surgical incision. Then the responses to identical PHE doses were measured again. Halothane did not influence the cardiovascular response to PHE: there was no dose-response shift for any cardiovascular variable. No arrhythmias or signs of ischaemia were observed. We conclude that one per cent halothane anaesthesia does not attenuate PHE-induced vasoconstriction in man.

Apomorphine-induced motor behavior in rats exposed prenatally to alcohol

We psychopharmacologically examined dopamine function in rats exposed to ethanol prenatally. Pregnant rats received liquid diets of 35% or 0% ethanol-derived calories (EDC), or ad lib lab chow (LC). Twenty-eight-day-old offspring received systemic doses of apomorphine chosen to stimulate predominantly presynaptic (0.02 or 0.1 mg/kg) or postsynaptic dopamine receptors (2.0 or 5.0 mg/kg). Behavior was scored automatically for 60 min in an “open field.” For males, prenatal ethanol exposure resulted in a dose-response shift to the left for locomotor activity. Females exposed to the liquid diet, with or without ethanol, showed less of an increase in locomotor activity following the 5.0 mg/kg dose of apomorphine than did LC controls. There were no effects of prenatal treatment on repetitious motor behavior in the automated “open field” or on stereotypy scored by direct observation in separate groups of rats. The results are consistent with an hypothesis that prenatal ethanol exposure alters the sensitivity of postsynaptic (perhaps mesolimbic) dopamine systems important to locomotor activity in young male rats.

Chemotactic activity of porcine insulin for human T lymphocytes in vitro.

T lymphocytes bear insulin receptors only after activation and entry into the cell cycle. To determine whether cell motility is concomitant with growth factor action in T lymphocytes, we measured the chemotactic activity of porcine insulin (10(-11) to 10(-5) M) for T lymphocytes. We found that the chemotactic response of human T cells activated with phytohemagglutinin (PHA) to porcine insulin was increased over that of resting T cells, with a concomitant two log leftward shift in the dose response. CD4+ and CD8+ subsets responded identically. Checkerboard analysis showed insulin to be chemotactic, as well as chemokinetic. The nature and time course of acquisition of the dose-response shift suggest that chemotaxis may be signaled by insulin acting on high affinity insulin receptors. The chemotactic effect of insulin exemplifies the general chemotactic effect of growth factors for motile target cells, and may be a useful model for the study of chemotactic signaling in T lymphocytes.

Dose-response shift in the ability of gilts to remain pregnant following exogenous estradiol-17 beta exposure.

Sixty mated gilts were assigned to a 2 X 6 factorial arrangement (n = 5) of day of injection (d 9 and 10 vs 12 and 13; d 0 = first day of estrus) and dose of estradiol-17 beta (0, .125, .5, 2, 8 and 32 mg X gilt-1 X d-1). Gilts were subsequently slaughtered on d 30; pregnancy was verified and percent embryonic survival calculated. A 64-fold shift in the dose-response curve for percent embryonic survival illustrated that the adverse effects of exogenous estradiol-17 beta were less when administered on d 12 and 13 as compared with d 9 and 10 (day X dose, P less than .01). This experiment demonstrated that the uterine-embryonic environment of d 12 and 13 pregnant gilts was more tolerant of exogenous estrogen alterations than that of d 9 and 10 pregnant gilts.