Experimental Biology and Medicine

Pharmacogenomics addresses the impacts of diverse and multiple genes in populations as determinants of responses of individual patients to drugs. The field has its roots in basic science, and is pivotal in drug development, elucidation of therapeutic efficacy, and constraining the risks of adverse drug reactions. Regulatory agencies are relying increasingly on pharmacogenomics for identification of patients who are particularly likely to benefit from treatment with specific agents and exclusion of those at risk of adverse drug reactions. Practical applications of pharmacogenomics already abound particularly in the use of drugs acting on the central nervous system and on the cardiovascular system. The Society for Experimental Biology and Medicine (SEBM) is proud and pleased to have devoted its 2008 symposium, presented at the annual Experimental Biology meeting in San Diego on April 6, 2008, to advances in pharmacogenomics with emphasis on drug development, regulatory agency considerations, and clinical applications.

The use of pharmacogenomics (PGx) today is almost ubiquitous in drug development and is advancing into the practice of medicine as an increasing number of drugs come to market with indications that are related to the presence or absence of a specific genetic biomarker. The authors review the history of PGx and its tools in research, in clinical trials and in clinical medicine. The economic, regulatory, and technological driving forces for adoption of PGx are then considered. Current impediments to a more robust proliferation of the benefits of these technologies are discussed—pharmaceutical companies, clinical education, required statistical methods, and intellectual property landscape.

This article discusses the current ambiguous state of federal regulatory agency control over pharmacogenomic testing, a subset of genetic testing that combines information about genetic variability with pharmacology in order to improve drug recommendations. An analysis of the common three terms used to evaluate regulation of pharmacogenomic testing: research validity, clinical validity, and clinical utility, followed by a case study involving U. S. Food and Drug Administration (FDA) regulation of laboratory developed tests, illustrates the present gap in pharmacogenomic oversight. The existing agency overlap in regulating pharmacogenomic testing leads to unclear or even contradictory authoritative advice.

Symptoms of central nervous system (CNS) disorders include abnormalities in both physical and psychological domains. Many drugs indicated for the treatment of CNS disorders are fraught with side effects and/or poor efficacy which impact patients’ quality of life and drives non-compliance. Moreover, for many CNS drugs such as antidepressants and antipsychotics, it takes time to determine whether a particular drug is efficacious in an individual patient. To optimize drug treatment for each patient, prescribing physicians often need to raise or lower doses, switch drug classes, or prescribe additional drugs to mitigate side effects, often in a “trial and error” fashion. Pharmacogenetic (PGx) testing, particularly in the realm of CNS therapy, can reduce the unpredictability of this process. By determining a patient’s genetic profile, individual therapy parameters may be predicted pre-treatment for drug efficacy, optimal drug dose, and the risk of adverse drug reactions (ADRs). The intent of this review is to highlight the power of PGx testing to predict the likelihood of ADRs and efficacy during the treatment of the following CNS disorders: epilepsy, bipolar disorder, schizophrenia and depression.

Hydroxyurea (HU) is an effective oral drug for the management of homozygous sickle cell anemia (SS) in part because it increases fetal hemoglobin (HbF) levels within sickle red blood cells (RBCs) and thus reduces sickling. However, results from the Multicenter Study of HU suggested that clinical symptoms often improved before a significant increase in HbF levels occurred. This indicated that HU may be acting through the modification of additional cellular mechanisms that are yet to be identified. Hence, in this study, we focused on the analysis of the sickle RBC membrane proteome +/− HU treatment. 2D-DIGE (Two Dimensional Difference In-Gel Electrophoresis) technology and tandem mass spectrometry has been used to determine quantitative differences between sickle cell membrane proteins in the presence and absence of a clinically relevant concentration of HU. In vitro protein profiling of 13 sickle RBC membrane samples +/− 50 μM HU identified 10 statistically significant protein spots. Of these, the most remarkable class of proteins to show a statistically significant increase was the anti-oxidant enzymes—catalase, thioredoxin peroxidase and biliver-din reductase and the chaperonin containing TCP1 complex assisting in the folding of RBC cytoskeletal proteins. Interestingly, catalase immunoblots showed an increase in the acidic forms of the enzyme within sickle RBC membranes on incubation with 50 μM HU. We further identified this modification in catalase to be phosphorylation and demonstrated that HU exposed SS RBC membranes showed a 2-fold increase in tyrosine phosphorylation of catalase as compared to counterparts not exposed to HU. These results present an attractive model for HU-induced post-translational modification and potential activation of catalase in mature sickle RBCs. These findings also identify protein targets of HU other than fetal hemoglobin and enhance the understanding of the drug mechanism.

Premature suture obliteration results in an inability of cranial and facial bones to grow, with craniofacial dysmorphology requiring surgical correction as a consequence. Understanding signaling pathways associated with suture morphogenesis might enable non-invasive treatment of patients with fused sutures. Tgf-β 2 induces premature suture fusion associated with increased cell proliferation both in vitro and in vivo. Tgf-β 2 and Egf signal transduction pathways use some signaling proteins in common to regulate proliferation and differentiation, leading to speculation that these two pathways converge to regulate normal suture development. It was therefore hypothesized that Egf could induce suture fusion, and that Tgf-β 2-induced suture closure occurred via an Egf-dependent pathway. A well-established fetal calvarial organ culture system was used to expose developing E19.5 fetal rat coronal sutures to Egf, Tgf-β 2 and SC-120, a blocker of Egf receptor activity. Co-culture experiments examined the effect of Egf on Tgf-β 2-induced suture closure when Egf was given either prior to or after Tgf-β 2 treatment. Histomorphometric measurement of suture width was done on sagittal sections through coronal sutures harvested after 5 days in culture. Western blotting using phospho-antibodies against Egf receptors was used to confirm Egf receptor activity. Suture width increased with increasing concentrations of Egf, demonstrating that Egf-induced cell activity alone was not sufficient to cause premature suture obliteration. Egf administered prior to Tgf-β 2 treatment rescued sutures from Tgf-β 2-induced suture obliteration, demonstrating that pre-exposure of cells to this powerful mitogen prevented their response to signals induced by Tgf-β 2. However, Egf added after Tgf-β 2 treatment had no effect on Tgf-β 2-induced suture closure. Blocking Egf activity after Tgf-β 2 treatment rescued sutures from Tgf-β 2-induced obliteration, indicating that Tgf-β 2 required Egf activity to induce suture obliteration. Appropriate timing of signal generation by Egf and Tgf-β 2 is critical for normal suture development and maintenance of suture patency.

Osteopontin (OPN) is a secreted, integrin-binding matrix phosphorylated glycoprotein that is overexpressed in many advanced cancers. However, the functional mechanisms by which OPN contributes to gastric cancer development are poorly understood. Here, we report that curcumin inhibited the growth of SGC7901 cell and induced apoptosis in a concentration- and time-dependent manner, while the acquired expression of OPN in SGC7901 cells dramatically promoted cell survival under serum depletion and prevented curcumin-induced apoptosis. Furthermore, PI3-K inhibitor LY294002 attenuated OPN-mediated Akt activation. Moreover, inhibiting the binding of OPN to α_vβ₃ integrins reduced activation of Akt. Taken together, these results demonstrate that the pro-survival and anti-apoptosis activities of OPN in gastric cancer cells are mediated in part through PI3-K/Akt pathway via α_vβ₃ integrins.

Obesity is associated with increased bone mineral density (BMD) but the mechanism for this is unclear. Serum levels of the adipokine adiponectin are inversely correlated with obesity, but results from studies on its relationship to bone mass are conflicting. The objective of this study was to compare bone mineral content (BMC), BMD and biomechanical strength properties of femur and lumbar vertebrae in 8- and 16-week old adiponectin transgenic mice (AdTg). These mice exhibit significantly elevated circulating adiponectin but have similar body weights compared to wild-type (WT) littermates that were used as controls. Female AdTg mice displayed significantly lower femur BMC at 8 and 16 weeks of age and femur neck peak load was significantly lower in 8-week old AdTg mice of both genders compared to controls. The peak load from compression testing of an individual lumbar vertebra was significantly lower in female AdTg mice compared to WT at 8 weeks, and this difference persisted at 16 weeks of age. In addition, lumbar vertebrae BMC was significantly lower in 16-week old male AdTg mice compared to WT although vertebra peak load was not different. Serum adiponectin levels were inversely correlated with femur BMC. In summary, elevated circulating adiponectin inhibits the acquisition of bone mass in growing mice and results in decreased biomechanical measures of functional strength that are surrogate measures of susceptibility to fractures. These results support a role for circulating adiponectin as a metabolic link that can explain, at least in part, the positive relationship between obesity and both bone mass and reduced susceptibility to fractures.

Doxorubicin (DOX) is an anthracycline antibiotic, and has been recognized as one of the most effective anti-neoplastic agents in cancer chemotherapy. However, its usefulness is limited by its profound cardiotoxicity. Licorice is one of the most frequently prescribed agents in traditional herbal medicine, and is also employed as a natural sweetening additive. In traditional Chinese medicine, licorice root is added to a variety of herbal preparations to detoxify the effects of the other herbs in the preparation. In the present study, we explored the possibility that Glycyrrhiza uralensis licorice may alleviate DOX-induced cardiotoxicity. The hexane/ethanol extract of Glycyrrhiza uralensis (HEGU), which lacks glycyrrhizin, was prepared because glycyrrhizin intake has previously been reported to induce hypertension. In an effort to determine whether HEGU ameliorates DOX-induced cytotoxicity in H9c2 rat cardiac myoblasts, the cells were pretreated with 0–15 mg/L HEGU, then treated with doxorubicin. The pretreatment of cells with HEGU resulted in a significant mitigation of DOX-induced reductions in cell numbers (34 ± 7%) and increases in apoptosis (53 ± 1%). The Western blot analysis of cell lysates showed that HEGU suppressed DOX-induced increases in the levels of p53, phospho-p53 (Ser 15), and Bax. In addition, HEGU induced an increase in the levels of Bcl-xL, regardless of DOX-treatment. HEGU inhibited the DOX-induced cleavage of caspases 9, 3, and 7, as well as DOX-induced poly(ADP-ribose) polymerase cleavage. Furthermore, HEGU caused reductions in the viable cell numbers of HT-29 human colon cancer cells (IC50 = 10.7 ± 0.3 mg/L), MDA-MB-231 human breast cancer cells (IC50 = 7.5 ± 0.1 mg/L), and DU145 human prostate cancer cells (IC50 = 4.7 ± 0.5 mg/L). HEGU augmented DOX-induced reductions in the viability of DU145 cells (15 ± 1%). These results indicate that HEGU may potentially be an effective agent for the alleviation of DOX-induced cardiotoxicity.

The blood-cerebrospinal fluid (CSF) barrier (BCB) resides within the choroid plexus, with the apical side facing the CSF and the basolateral side towards the blood. Previous studies demonstrate that manganese (Mn) exposure in rats disrupts iron (Fe) homeostasis in the blood and CSF. The present study used a primary culture of rat choroidal epithelial cells grown in the two-chamber Transwell system to investigate the transepithelial transport of Fe across the BCB. Free, unbound Fe as [⁵⁹Fe] was added to the donor chamber and the radioactivity in the acceptor chamber was quantified to determine the direction of Fe fluxes. Under the normal condition, the [⁵⁹Fe] efflux (from the CSF to the blood) was 128% higher than that of the influx (P < 0.01). Mn exposure significantly increased the efflux rate of [⁵⁹Fe] (P < 0.01) and the effect was inhibited when the cells were pre-incubated with the antibody against divalent metal transport 1 (DMT1). Moreover, when the siRNA knocked down the cellular DMT1 expression, the elevated Fe uptake caused by Mn exposure in the choroidal epithelial Z310 cells was completely abolished, indicating that Mn may facilitate Fe efflux via a DMT1-mediated transport mechanism. In vivo subchronic exposure to Mn in rats reduced Fe clearance from the CSF, as demonstrated by the ventriculo-cisternal brain perfusion, along with up-regulated mRNAs encoding DMT1 and transferrin receptor (TfR) in the same animals. Taken together, these data suggest that free Fe appears to be favorably transported from the CSF toward the blood by DMT1 and this process can be facilitated by Mn exposure. Enhanced TfR-mediated influx of Fe from the blood and ferroportin-mediated expelling Fe toward the CSF may compromise DMT1-mediated efflux, leading to an increased Fe concentration in the CSF as seen in Mn-exposed animals.

Ritonavir, a protease inhibitor used in combination antiretroviral therapy for HIV-1 infection, is associated with an increased risk of premature atherosclerosis. The aim of the present study was to assess the effects of ritonavir, in the absence of added lipoproteins, on the expression of genes that control cholesterol trafficking in human monocytes/macrophages. Design: THP-1 cells were used to study the effects of ritonavir on the expression of CD36, ATP binding cassette transporters A1 (ABCA1) and G1 (ABCG1), scavenger receptor B class I (SR-BI), caveolin-1 and sterol 27-hydroxylase (CYP27). Exposure to ritonavir (2.5 μg/ml) increased CD36 protein (28%, P < 0.05) and mRNA (38%, P < 0.05) in differentiated THP-1 macrophages, but not in undifferentiated monocytes. This effect was not related to the increase in PPARγ expression (51%, P < 0.05) caused by ritonavir. Ritonavir also reduced SR-BI protein levels (46%, P < 0.05) and increased CYP27 (43%, P < 0.05) and ABCA1 (49%, P < 0.05) mRNA expression. Liver X receptor α (LXRα) mRNA, protein and binding activity were also increased by ritonavir treatment. Conclusions: We propose that ritonavir induces ABCA1 expression in THP-1 macrophages through LXRα. The increase in ABCA1 and other cholesterol efflux mediators, such as CYP27, may compensate CD36 induction. Therefore, we suggest that the net effect of ritonavir on macrophages in the absence of lipoproteins is not clearly proatherogenic.

Despite intensive research as to the pathogenesis of lipopolysaccharide (LPS)-related inflammation with coagulatory disturbance, their exacerbating factors have not been well explored. This study examined the effects of pulmonary exposure to two types of nano-sized materials (carbon nano-tubes: CNT [single-wall: SWCNT, and multi-wall: MWCNT]) on lung inflammation and consequent systemic inflammation with coagulatory disturbance induced by pulmonary exposure to LPS in mice and their cellular mechanisms in vitro. ICR male mice were divided into 6 experimental groups that intra-tracheally received the vehicle, two types of CNT (4 mg/kg), LPS (33 μ g/kg), or LPS plus either type of CNT. Twenty-four hours after treatment, both types of CNT alone induced lung inflammation with enhanced lung expression of proinflammatory cytokines, but did not synergistically exacerbate lung inflammation elicited by LPS. SWCNT significantly induced/ enhanced pulmonary permeability and hyperfibrinogenemia and reduced activated protein C in the absence or presence of LPS, whereas MWCNT did moderately. Both CNT moderately, but not significantly, elevated circulatory levels of proinflammatory cytokines and chemokines. In the presence of LPS, CNT tended to elevate the levels of the mediators with an overall trend, which was more prominent with SWCNT than with MWCNT. In vitro study showed that both CNT amplified LPS-induced cytokine production from peripheral blood monocytes. These results suggest that CNT can facilitate systemic inflammation with coagulatory disturbance, at least in part, via the activation of mononuclear cells, which is accompanied by moderate enhancement of acute lung inflammation related to LPS.

The chemical structure of the main fluorescenting compound in the ethanolic extract (mother tincture) of the American yellow jasmine, Gelsemium sempervirens, was determined by employing ¹H nuclear magnetic resonance (NMR), ¹³C NMR, mass spectroscopy, high-performance liquid chromatography (HPLC), correlation spectroscopy (COSY), and Fourier transform infrared (FTIR) spectroscopy analyses. Spectrofluorometric analysis has been made of the mother tincture and its agitated serial dilutions (up to 12th potency) prepared according to a homeopathic procedure in which serial, agitated dilutions were made separately in glass and polypropylene containers. The succussions were made by employing three different modes: hand jerk, sonication, and vortexing. The chemical formula of scopoletin, the main fluorescent compound, was determined to be C₁₀H₈O₄ having a molecular weight of 192.17. Significant differences were noted between the remedies prepared in the two types of containers. Further, a comparison between any two methods of agitation revealed significant differences in fluorometric data of remedies at certain potency levels. The biological (anticancer) action of the crude extract, the alkaloid scopoletin, and 2C potency of Gelsemium sp were tested in vitro on the HeLa cell line through fluorescence microscopy, the 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and fluorescent activated cell sorting (FACS). The role of nanoparticles presumably derived from the containers, their orientation, and their interaction with the starting substance during the dynamization process initiated by different modes of agitation could possibly be attributed to the differences noted in the fluorometric data of potencies prepared in the two types of containers and among the three different means of succussion tested.

To examine the reaction of tumour arteries to endothelin-1, we obtained arteries supplying blood flow to colorectal tumours from patients, as well as mesenteric arteries supplying the normal colon tissue from the same patients and mesenteric arteries from patients without a colorectal tumour pathology. The contraction in response to endothelin-1 and the relaxation produced by bradykinin was recorded in each of these arteries. Accordingly, the sensitivity to endothelin-1 but not the maximal response, was higher in the arteries supplying colorectal tumours than in mesenteric arteries supplying normal colon or in mesenteric arteries from patients with no tumour pathology. The contraction produced by endothelin-1 was not modified by exposure to L-NAME or meclofenamate in arteries supplying both the tumour and the normal colon. The endothelin ET_A andET_B receptors were expressed similarly in arteries supplying the tumour or normal colon. However, the antagonist of the endothelin ET_B receptors BQ788 (10⁻⁶ M) decreased the contractions in the arteries supplying the tumour but not in those supplying the normal colon. By contrast, the antagonist of endothelin ET_A receptors BQ123 (10⁻⁶ M) reduced the contraction equally in both these types of arteries. Likewise, in arteries precontracted with U46619, the relaxation in response to bradykinin was similar in all three types of arteries. Together, these results suggest that the arteries supplying human colorectal tumours are more sensitive to endothelin-1, which could be due to the enhanced activity of endothelin ET_B receptors in the absence of any change in the modulatory effect of nitric oxide or prostanoids in the arterial response to this peptide.

Genetic and environmental factors are involved in prostate cancer (PCa) etiology. Single nucleotide polymorphisms (SNPs) may contribute to the PCa pathogenesis. The goal of this study is to determine the role of vitamin D receptor (VDR) gene polymorphisms and haplotypes in the development and progression of sporadic PCa. One hundred and thirty-three PCa patients and 157 age-matched healthy controls were genotyped for the Apa I (rs7975232), Bsm I (rs1544410) and Taq I (rs731236) polymorphisms in VDR gene by using polymerase chain reaction-restriction fragment length polymorphism. An association was observed between the Apa I polymorphism and PCa predisposition (P = 0.03). When compared with AA genotype, there was a highly notable difference in the frequencies of the Aa (P = 0.02), aa (P = 0.026) and Apa I ‘‘a’’ allele carriers (Aa + aa) (P = 0.009) genotypes. Furthermore, we found a statistical difference in the allele frequencies of the Apa I polymorphism between the sporadic PCa patients and control subjects (P = 0.013). The genotype distribution for the Bsm I and Taq I polymorphisms were similar between cases and controls (P > 0.05). No clinically significant relationship was found between the three-locus haplotypes and development of sporadic PCa. The genotype frequencies for the three polymorphisms of the VDR gene within subgroups of PCa (defined by tumor stage, Gleason score, PSA levels) were also analyzed, but no statistically noteworthy difference was observed (P > 0.05). As far as we know, this is the first study which investigates the relationship between VDR genotypes and sporadic PCa in the Turkish population. Our findings suggest that the VDR ApaI (rs7975232) polymorphism may play a role in the development of sporadic PCa.