Urethane Administration Research Articles

Acute hyperosmotic stimulus of the Organum Vasculosum of the Lamina Terminalis leads to increase in the blood pressure dependent on release of ATP into the Paraventricular Nucleus of the Hypothalamus of rats

The neural network involved in hyperosmolality-induced hypertension includes the activation of the organum vasculosum of the lamina terminalis (OVLT) with monosynaptic connections to the paraventricular nucleus of the hypothalamus (PVN) that expresses an abundance of purinergic receptors including P2 receptors. Previously, we have shown that hyperosmotic-induced sympathoexcitation at the level of the PVN depends on the P2 receptors. More recently, we have shown that hyperosmotic stimulus induces the release of ATP in the PVN, at least in part, from astrocytes. Here we hypothesize that the increase in the blood pressure (BP) induced by acute hyperosmotic stimulus in the OVLT involves the release of ATP within the PVN neurons, acting on P2 receptors. Thus, we sought to determine (1) whether P2X7-expressing OVLT and PVN neurons are activated by hyperosmotic challenge, and (2) what effects P2 receptor antagonism on changes in the BP mediated by an acute hyperosmotic stimulus in the OVLT. Adult Wistar rats (10-12 weeks old; CEUA ICB/USP: #4895140819 and #1521230221) were used in two protocols: 1) Double-labeling immunofluorescence of P2X7 (anti-P2X7 receptor, 1:500) expressing neurons with FOS (anti-cfos, 1:3000) animals received i.v administration of hypertonic (NaCl, 3M, n=4) or isotonic saline (NaCl, 0.154M, n=4), followed by evaluation of immunostaining to FOS and P2X7 receptors in the OVLT and PVN; 2) OVLT-PVN microinjections and BP monitoring: under anesthesia rats (n=5), received catheters in the femoral vein and artery for the administration of urethane (1g/kg) and BP recording signals, respectively. Microinjections of 3M NaCl (50nL) were administered into the OVLT, before and after bilateral antagonism of P2 purinergic receptors (PPADS, 10uM-100nL on each side) in the PVN with simultaneous BP recording. Data were expressed as the mean ± SD, and statistical significance level of p<0.05 by Student’s T-test. Immunoreactive neurons for the P2X7 receptor subunit were observed in all compartments of the OVLT and PVN. Hyperosmotic stimulus significantly increased the number of Fos+ neurons in both the OVLT and PVN. While a small number of P2X7 receptors expressing OVLT and PVN neurons was observed in rats treated with isotonic saline, significantly more P2X7-expressing neurons expressed Fos+ in the OVLT with hypertonic saline. The stimulus of the OVLT with 3M NaCl elicited a significant increase in the BP. This effect was attenuated after bilateral injection of PPADS in the PVN (ΔMAP: NaCl OVLT:+10±1mmHg vs. NaCl OVLT post-PPADS PVN: +2.4±1.3mmHg, p<0.0001). Collectively, our findings indicate that hyperosmotic stimulation activates a subset of P2X7 expressing OVLT and PVN neurons and that the elevation in the BP induced by hyperosmotic stimulus in the OVLT involves the endogenous release of ATP within the PVN, likely as part of the neural control of circulation during osmotic challenges. Financial Support: FAPESP #2019/19894-8, CAPES #88887.364432/2019-00. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Changes in the Cellular Composition of the Rat Thymus 90 Days After Urethane Injection Against the Background of Selenium Intake

The aim of the study is to assess the cellular composition of rat thymus when isolated administration of selenium, urethane and combined exposure to two factors. Material and methods. The study was performed on 34 male Wistar rats, which were divided into 4 groups: 1 – intact, 2 – rats, which received selenium only for 1 month, 3 – animals, which were injected once intraperitoneal urethane, 4 – rats with combined selenium and urethane action. Histological preparations of the thymus were prepared according to generally accepted methods and further evaluated by the method of review microscopy, morphometry, immunohistochemical reactions, electron microscopy, statistical processing of data. Results. Ninety days after selenium, the rat thymus structure is slightly different from intact animals. The introduction of urethane leads to the development of excitation involution of the thymus, which is characterized by morphological changes, and manifests itself in a change in the shape of the organ lobes, in the expansion of the intercole gaps and in the fat tissue thereof, hyperplasia of the cortical substance of the thymus by increasing the number of mature lymphocytes, epithelial cells positive to pancytokeratin. At the ultramicroscopic level, the thymus lymphocyte decreases its nuclear-cytoplasmic index, and increases the number of mitochondria that have a light matrix with a small amount of crystal. Apoptotic bodies and a large number of electron-transparent vacuoles are visualized. When combined, the thymus structure differs from the intact group by an enlarged brain substance, but the number of epithelial cells that are positive to pancytokeratin and Ki67+-cells, as well as cells that express apoptosisregulating protein, decreases. Electron microscopy also indicates a large amount of mitochondria, but with welldefined crystals, when combined with selenium and urethane. There are a small number of electron-transparent vacuoles. Conclusion. The obtained data make it possible to draw a conclusion about the positive influence of the exchange rate of selenium on the T-cell link of immunity, at least for this period of study. In the course of the experiment, it was found that, against the background of the development of the adenoma of the lung caused by the introduction of urethane, at the stage of alveolar epithelium dysplasia signs of excitant thymus involution were detected. When selenium and urethane are combined, less pronounced changes are observed in the examined organ than in the isolated administration of urethane.

EGCG alleviates obesity-exacerbated lung cancer progression by STAT1/SLC7A11 pathway and gut microbiota

Leptin is a nutritional cytokine, and it is closely related to the progression of cancer. However, the detailed effect of leptin in lung cancer remains poorly known. We found leptin-induced A549 cell proliferation, migration, and invasion, which was reversed by epigallocatechin gallate (EGCG) from green tea. Currently, we found that leptin-triggered M2 polarization of tumor-associated macrophages was inhibited by EGCG. Then, to investigate the underlying mechanism effect of leptin on A549 cells was studied. Aberrant activities of STAT1 are implicated in cancer development. Based on the cancer genome atlas data, STAT1 acted as an oncogene in lung cancer and EGCG greatly reduced STAT1 expression in A549 cells. Ferroptosis is an iron-dependent nonapoptotic cell death. STAT1 served as a transcriptional activator for SLC7A11. EGCG restrained lung cancer cell growth induced by leptin via targeting STAT1-SLC7A11 mediated ferroptosis. A high-fat diet (HFD) feeding condition was combined with a multi-dose urethane-induced lung tumorigenesis model using C57BL/6J mice. Obesity was induced with a 60 kcal% HFD feeding. Serum leptin levels increased in urethane-administered and HFD-fed mice. Compared to the control diet-fed mice, the HFD-fed mice exhibited increased lung tumor burden and typical pro-tumorigenic STAT1 activation in lung tissues after urethane administration. In addition, HFD alters the gut microbiome by decreasing the abundance of Clostridia and by increasing the abundance of Deltaproteobacteria and Epsilonproteobacteria while EGCG exhibited a reversed effect. These findings suggested that leptin promoted the development of lung tumorigenesis in vitro and in vivo via mediating activation of the STAT-SLC7A11 pathway and gut microbiota.

Targeted Imaging of Lung Cancer with Hyperpolarized 129Xe MRI Using Surface-Modified Iron Oxide Nanoparticles as Molecular Contrast Agents.

Hyperpolarized 129Xe (HP 129Xe) MRI enables functional imaging of various lung diseases but has been scarcely applied to lung cancer imaging. The aim of this study is to investigate the feasibility of targeted imaging of lung cancer with HP 129Xe MRI using surface-modified iron oxide nanoparticles (IONPs) as molecular targeting contrast agents. A mouse model of lung cancer (LC) was induced in nine mice by intra-peritoneal injection of urethane. Three months after the urethane administration, the mice underwent lung imaging with HP 129Xe MRI at baseline (0 h). Subsequently, the LC group was divided into two sub-groups: mice administered with polyethylene glycol-coated IONPs (PEG-IONPs, n = 4) and folate-conjugated dextran-coated IONPs (FA@Dex-IONPs, n = 5). The mice were imaged at 3, 6, and 24 h after the intravenous injection of IONPs. FA@Dex-IONPs mice showed a 25% reduction in average signal intensity at cancer sites at 3 h post injection, and a 24% reduction at 24 h post injection. On the other hand, in PEG-IONPs mice, while a signal reduction of approximately 28% was observed at cancer sites at 3 to 6 h post injection, the signal intensity was unchanged from that of the baseline at 24 h. Proton MRI of LC mice (n = 3) was able to detect cancer five months after urethane administration, i.e., later than HP 129Xe MRI (3 months). Furthermore, a significant decrease in averaged 1H T2 values at cancer sites was observed at only 6 h post injection of FA@Dex-IONPs (p < 0.05). As such, the targeted delivery of IONPs to cancer tissue was successfully imaged with HP 129Xe MRI, and their surface modification with folate likely has a high affinity with LC, which causes overexpression of folate receptors.

Therapeutic potential of human serum albumin nanoparticles encapsulated actinonin in murine model of lung adenocarcinoma

Non-small cell lung cancer comprises 85% of the global lung cancer cases. Conventional chemotherapeutics possess certain limitations like systemic toxicity and drug resistance that requires the development of new therapeutic agents for successful treatment of lung cancer. Actinonin, a human peptide deformylase inhibitor, has demonstrated anti-cancerous properties in various leukemias and solid cancer types. However, it has limited therapeutic application because of its low bioavailability and systemic toxicity if administered in free form. This limitation can be overcome by using nano-delivery systems that will increase the therapeutic efficacy of actinonin. In the present study, human serum albumin actinonin nanoparticles were prepared using a desolvation technique and folic acid was conjugated to lysine residues of albumin for effective delivery to the lung. The lung adenocarcinoma model was established 24 weeks after intraperitoneal administration of urethane and chemotherapeutic efficacy of free as well as nanoencapsulated actinonin was evaluated. This study demonstrated anti-proliferative potential of folic acid conjugated human serum albumin nanoparticles encapsulating actinonin. The intraperitoneally administered nanoformulation exhibited sustain release profile of actinonin with longer half-life and mean retention time. The reduced dose frequency resulted in therapeutic efficacy comparable to free drug in vivo in terms of 100% survival and reduced tumor burden along with downregulation of epidermal growth factor receptor, folate receptor α and peptide deformylase expression in lung adenocarcinoma mice model. Therefore, actinonin encapsulated albumin nanoparticles-based therapy holds great potential as an alternative strategy to improve its anti-cancerous activity against lung adenocarcinoma.

Novel therapeutic modalities target cell signaling of Renin-Angiotensinsystem/NF-κB-induced cell cycle arrest and apoptosis in urethane-induced lung cancer in mice: An in vivo study.

Lung cancer has risen to the top of the list of cancer-related deaths worldwide. Aliskiren is a direct renin inhibitor. This study aims to investigate the impact of cell signaling ofRenin-Angiotensinsystem(RAS)/NF-κB on lung cancer by investigating the potential therapeutic effects of aliskiren for lung cancer treatment in urethane-induced lung cancer in mice. Male BALB/c mice were randomly assigned to one of five treatment groups for 150 days, including (1) normal control; (2) aliskiren (25 mg/kg/i.p) daily, (3) urethane at a dose of 1.5 g/kg (i.p) at Day 1 and 60 (nonsmall cell lung cancer[NSCLC] group) (4) NSCLC mice received carboplatin (15 mg/kg/i.p) every other day for the last 4 successive weeks and (5) NSCLC mice treated with aliskiren daily. Tumor size was determined based on blood sampling, and lungs were isolated for biochemical analysis, western blot analysis assay, and histopathological examination. Urethane demonstrated significant changes in all biochemical and molecular parameters and histological patterns. Aliskiren-treated mice had significantly lower levels of NF-κB p65, Bcl-2, cyclin D1, ICAM-1, MMP-2, and Nrf2, with an increase in the catalytic activity of caspase-3 due to its RAS inhibitory mechanism. The combined urethane administration with aliskiren demonstrated a significant improvement in the histopathological examination. RAS/NF-B cell signaling is a potential therapeutic target for preventing and treating lung adenocarcinoma, evidenced by the fundamental cytotoxic mechanism and attenuation of metastasis and angiogenesis induced by the treatment of NSCLC mice with aliskiren.

The Sulfiredoxin‐Peroxiredoxin axis promotes urethane‐induced lung adenocarcinoma through the regulation of the tumor microenvironment

Lung adenocarcinoma is the most common type of lung cancer and has a strong association with tobacco smoking. Smoking leads to the accumulation of reactive oxygen species (ROS) and oxidative stress damages to macromolecules, leading to abnormal cell growth & proliferation, transformation, changes in cell metabolism and a variety of other physiological functions. Accumulation of ROS only leads to increased expression of cellular antioxidants that contribute to tumorigenesis and cancer progression. Sulfiredoxin (Srx) is the only enzyme that reduces over‐oxidized forms of typical 2‐Cys Peroxiredoxins (Prxs). Compared to other Prx family members, Srx preferentially interacts with and has a higher binding affinity to Prx4. Our previously work demonstrated that the Srx‐Prx4 axis enhances the RAS/RAF‐MEK‐ERK signaling cascade to promote lung cancer cell proliferation and metastasis. However, the role of the Srx‐Prx axis in de novo lung tumorigenesis has not been explored.In this study, Prx4‐/‐ mice and Srx‐/‐Prx4‐/‐ mice were generated and mouse lung carcinogenesis was induced by a well‐established urethane protocol. Briefly, wildtype and knockout mice at 8‐week of age were injected intraperitoneally with 1g/kg of urethane once per week for three successive weeks. Ten weeks after the administration of urethane, all mice were euthanized and examined for tumors. Our results indicate that both knockout mice have significantly reduced rates on tumor incidence, multiplicity, and size. Compared with tumors from wildtype mice, the rate of cell proliferation in tumors from either knockout mice is significantly decreased.Macrophages are the most abundant immune cells identified in the tumor microenvironment of solid tumors and their presence correlates with reduced survival in most cancers. In addition to all other markers, we also compared the presence of two distinct states of polarized macrophages including the classically activated macrophage (M1) and the alternatively activated macrophage subsets (M2) in urethane‐induced tumors using specific surface marker staining. We found that there are significantly increased numbers of M1 cells in tumors of either Prx4‐/‐ or Srx‐/‐Prx4‐/‐ mice compared with those of wildtype mice. In contrast, there are significantly few numbers of M2 cells in tumors from either knockout mice.It has been shown that M1 macrophages have immune‐stimulatory properties through the expression of a series of pro‐inflammatory cytokines, chemokines, and effector molecules, whereas M2 cells express a wide array of anti‐inflammatory molecules. Therefore, the difference in tumor‐associated macrophages caused by the loss of Prx4 or Srx and Prx4 may contribute to the reduced lung tumorigenesis observed in the mouse model. In addition, bioinformatic analysis shows that Prx4 is overexpressed in the majority of cancers, and the increased expression of Srx and Prx4 both show a strong correlation with poor prognosis in lung adenocarcinoma patients.

High-Fat Diet-Related Obesity Promotes Urethane-Induced Lung Tumorigenesis in C57BL/6J Mice.

Epidemiological studies have recently shown that obesity increases lung cancer risk, but the underlying biological connection is unclear. To determine whether high-fat diet (HFD)-induced obesity influences the susceptibility to chemical-induced lung tumorigenesis, a HFD feeding condition was combined with a multi-dose urethane-induced lung tumorigenesis model using C57BL/6J mice. In cell culture models, lung cancer cell lines A549 and H460 were used to investigate the effect of leptin on cell viability and its underlying mechanism of action. The results showed that obesity was induced with a 60 kcal% HFD feeding. Serum leptin levels increased with HFD feeding and further increased in urethane-administered and HFD-fed mice. Compared to the control diet-fed mice, the HFD-fed mice exhibited increased lung tumor burden and typical pro-tumorigenic STAT3 pathway activation in lung tissues after urethane administration. In vitro, leptin significantly increased the viability of lung cancer cell lines A549 and H460 in a dose-dependent manner by activation of STAT3, Bcl-2, and cyclin D1. These effects were significantly attenuated when PI3K or mTOR were inhibited by LY294002 or rapamycin, respectively. These results suggested that HFD-induced obesity could promote the development of lung tumorigenesis in C57BL/6J mice, and leptin-mediated activation of the PI3K/Akt/mTOR/STAT3 pathway was likely involved in this mechanism.

Urethane attenuates early neuropathology of diisopropylfluorophosphate-induced status epilepticus in rats

Seizures can be evident within minutes of exposure to an organophosphorus (OP) agent and often progress to status epilepticus (SE) resulting in a high mortality if left untreated. Effective medical countermeasures are necessary to sustain patients suffering from OP poisoning and to mitigate the ensuing brain injury. Here, the hypothesis was tested that a single subanesthetic dose of urethane prevents neuropathology measured 24 h following diisopropylfluorophosphate (DFP)-induced SE. Adult Sprague-Dawley rats were injected with DFP to induce SE. During SE rats displayed increased neuronal activity in the hippocampus and an upregulation of immediate early genes as well as pro-inflammatory mediators. In additional experiments rats were administered diazepam (10 mg/kg, ip) or urethane (0.8 g/kg, sc) 1 h after DFP-induced SE and compared to rats that experienced uninterrupted SE. Cortical electroencephalography (EEG) and power analysis strengthen the conclusion that urethane effectively terminates SE and prevents the overnight return of seizure activity. Neurodegeneration in limbic brain regions and the seizure-induced upregulation of key inflammatory mediators present 24 h after DFP-induced SE were strongly attenuated by administration of urethane. A trivial explanation for these beneficial effects, that urethane simply reactivates acetylcholinesterase, has been ruled out. These findings indicate that, by contrast to rats administered diazepam or rats that experience uninterrupted SE, the early neuropathology after SE is prevented by subanesthetic urethane, which terminates rather than interrupts, SE.

MafB silencing in macrophages does not influence the initiation and growth of lung cancer induced by urethane.

An increased number of tumor-associated macrophages (TAMs) that exhibit the M2 macrophage phenotype is related to poorer prognosis in cancer patients. MafB is a transcription factor regulating the differentiation of macrophages. However, involvement of MafB for the development of TAMs is unknown. This study was designed to investigate the role of MafB in a murine urethane-induced lung cancer model. Urethane was injected intraperitoneally into wild-type and dominant-negative MafB transgenic mice. Twenty-four weeks later, mice were sacrificed and their lungs removed for pathological analysis. The numbers and mean areas of lung cancer were evaluated. In addition, the numbers of Mac-3-positive macrophages were evaluated in each tumor. The numbers and mean areas of lung cancer induced by urethane administration were not significantly different between wild-type and dominant-negative MafB transgenic mice. The numbers of TAMs in lung cancer tissue were not significantly different between the two groups. MafB silencing using dominant-negative MafB did not influence the initiation and growth of lung cancer in mice exposed to urethane. These data suggest that MafB may not be related to the development of TAMs.

Abstract 1673: Alternative NF-κB signaling promotes inflammatory cell recruitment and lung tumor formation

Abstract Nuclear factor-κB (NF-κB) is a transcription factor that can be activated through either the classical or alternative signaling pathways. In lung tumors, high levels of classical and alternative pathway components have been observed. While many studies have demonstrated that classical NF-κB signaling is a central pathway regulating inflammation and tumorigenesis, the role of alternative NF-κB signaling remains undefined. We hypothesize that alternative NF-κB signaling in the lung epithelium plays a key role in inflammation and lung tumor formation through paracrine signaling to the inflammatory microenvironment. To investigate alternative NF-κB signaling in the lung epithelium, we generated a transgenic mouse model with doxycycline (dox)-inducible expression of the alternative pathway component p52 in airway epithelial cells (designated ALTA for alternative pathway trans-activated). When placed on dox, nuclear p52 is increased in the lungs of ALTA mice with no effect on activation of other NF-κB components. Surprisingly, alternative pathway activation alone did not cause a significant increase in inflammatory cell recruitment. However, in conjunction with the inflammatory stimulus lipopolysaccharide (LPS), alternative pathway activation caused increased mortality, which was associated with a significant increase in inflammation and increased lung permeability. To determine the effect of alternative pathway activation on lung tumor formation, mice were injected with the lung carcinogen urethane. Six weeks after urethane administration, ALTA mice demonstrated an increased number of bronchoalveolar lavage neutrophils, and by 6 months post-urethane, ALTA mice had an increased number of tumors, increased tumor size, and more advanced lesions. Together, these data suggest that alternative NF-κB signaling plays an important role in stimulus-induced inflammatory cell recruitment and that alternative pathway activation can promote lung tumor formation and progression. By defining the role of alternative NF-κB signaling in tumor formation, we hope to gain insight into the role of inflammation in lung carcinogenesis and to identify novel pathways and signaling targets that could potentially serve as therapeutic targets. Citation Format: Jamie Alicyn Ausborn, Dong-Sheng Cheng, Vasiliy Polosukhin, Wei Han, Fiona Yull, Timothy Blackwell. Alternative NF-κB signaling promotes inflammatory cell recruitment and lung tumor formation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1673. doi:10.1158/1538-7445.AM2014-1673

Cav2.3 E-/R-type voltage-gated calcium channels modulate sleep in mice

Mammalian sleep is characterized by cycles of REM and non-REM (NREM), i.e. slow-wave sleep (SWS) phases. The major neuroanatomical basis of SWS is the thalamocortical circuitry, which operates in different functional modes to determine the state of vigilance. At high vigilance, the tonic mode predominates; stages of low vigilance and SWS are characterized by rebound burst firing. Electrophysiologically, rebound bursting depends on low-threshold Ca2 + spikes and T-type Ca2 + channels have been shown to modulate SWS. We recently demonstrated that Cav2.3 R-type Ca2 + channels are capable of modulating absence seizures, a pathophysiological aberration of the thalamocortical oscillations related to SWS. We thus analyzed sleep architecture in control and Cav2.3(−|−) mice using implantable electroencephalography (EEG)/electromyography (EMG) radiotelemetry during spontaneous and urethane-induced sleep. The results demonstrate significantly reduced total sleep time and impairment of SWS generation in Cav2.3(−|−) mice, which affects global sleep architecture (i.e. the ratio of REM to NREM). Furthermore, the relative δ power is significantly reduced in Cav2.3(−|−) mice during NREM sleep although these mice display longer prior wakefulness, possibly indicating disturbances in sleep homeostasis. This observation is supported by recordings following urethane administration. This is the first study to shed light on the fundamental role of Cav2.3 channels in rodent sleep physiology.

Nrf2 Prevents Initiation but Accelerates Progression through the Kras Signaling Pathway during Lung Carcinogenesis

Nrf2 (Nfe2l2) governs cellular defenses against oxidative and electrophilic stresses and protects against chemical carcinogenesis. However, many cancers have been found to accumulate NRF2 protein, raising questions of precisely how Nrf2 contributes to carcinogenesis. In this report, we explored such questions in an established urethane-induced multistep model of lung carcinogenesis. Consistent with earlier observations, Nrf2-deficient (Nrf2(-/-)) mice exhibited a relative increase in tumor foci by 8 weeks after urethane administration. However, after 16 weeks, we observed a relative reduction in the number of tumors with more malignant characteristics in Nrf2(-/-) mice. Furthermore, all Nrf2(+/+) tumors harbored activated mutations in Kras, whereas Nrf2(-/-) tumors were rarely associated with similar Kras mutations. Overall, our results established that Nrf2 has two roles during carcinogenesis, one of which is preventive during tumor initiation and the second that promotes malignant progression. These findings establish Nrf2 inhibitors as rational tools to prevent malignant progression in lung cancer, whereas Nrf2 activators are more suited for lung cancer prevention.

The mitochondrial dysfunction plays an important role in urethane-induced lung carcinogenesis

Mitochondrial dysfunction is an important factor as genetic change in controlling the growth of tumors. The current study explored whether mitochondrial dysfunction correlated with urethane-induced lung carcinogenesis in BALB/c and C57BL/6 mice that were given single- or multi-dose intraperitoneal injections of urethane. We found that mice susceptible to lung tumor formation displayed a rapid increase in the respiratory control ratio in lung mitochondria after urethane exposure, whereas resistant mice that failed to develop lung tumors maintained the same respiratory control ratio as normal, untreated mice. Furthermore, repeated urethane administration or continuous 2,4-dinitrophenol (an uncoupling agent of oxidative phosphorylation) treatment, following a single urethane exposure, could overcome resistance to carcinogenesis. In contrast, multi-dose urethane-treated mice that received genipin (a highly selective inhibitor of uncoupling protein 2) following their first dose of urethane showed a lower tumor incidence. In addition, a higher uncoupling protein 2 level and a lower complex IV level in the lungs correlated with subsequent tumor formation in BALB/c and C57BL/6 mice. In vitro, urethane suppressed cell proliferation and induced soft-agar colonies in parental L929 cells with complete mitochondrial DNA but not in ρ° L929 cells lacking mitochondrial DNA. These studies suggest that abrogation of mitochondrial is essential during urethane induced lung carcinogenesis and that uncoupling inhibition can reverse cancer morphogenesis, thus presenting an appealing method to prevent lung carcinogenesis.

Reduction in the Number of Animals and the Evaluation Period for the Positive Control Group in Tg.rasH2 Short-Term Carcinogenicity Studies

The lack of a clear guidance on the adequate number of animals used for positive controls in the short-term (26-weeks) transgenic mouse carcinogenicity studies has resulted in the use of high number of animals. In our earlier Tg.rasH2 studies, 25 mice/sex were used in the urethane-positive control dose groups that were sacrificed by 18 weeks. Based on a robust response, several of our protocols for Tg.rasH2 studies with 15 mice/sex and terminal sacrifice at 17 ± 1 weeks were submitted and accepted by the Carcinogenicity Assessment Committee of the US Food and Drug Administration since we demonstrated close to 100% response for the development of lung and splenic tumors (target organs) in 500 mice/sex. These 500 mice/sex included 17 groups of 25 mice/sex and 5 groups of 15 mice/sex. The objective of this investigation was to determine whether the number of animals can be further reduced along with the shortened duration of exposure to urethane. Accordingly, 10 Tg.rasH2 mice/sex/group were administered a total of 3 intraperitoneal (IP) injections of urethane (1000 mg/kg per day) on study days 1, 3, and 5, and the presence of tumors in the lungs and spleen was evaluated after 8, 10, 12, 14, or 16 weeks. Our results demonstrate that 100% of the mice at 8 weeks had developed lung tumors, whereas close to 100% of the mice at 14 weeks had developed splenic tumors. Based on the development of lung tumors alone in 100% of the mice, we recommend that 10 mice/sex are sufficient and that these mice can also be sacrificed as early as 10 ± 1 weeks following the administration of urethane.

Endothelial Cell–Specific Deletion of Transcription Factor FoxM1 Increases Urethane-Induced Lung Carcinogenesis

Vascular endothelial cells provide essential support to the tumor microenvironment, but little is known about the transcriptional control of endothelial functions during tumorigenesis. Here we define a critical role for the Forkhead transcription factor FoxM1 in modulating the development of tumor-associated endothelial cells. Pulmonary tumorigenesis induced by urethane administration was compared in mice genetically deleted for FoxM1 in endothelial cells (enFoxm1(-/-) mice). Notably, lung tumor number and size were increased in enFoxm1(-/-) mice. Increased tumorigenesis was associated with increased proliferation of tumor cells and increased expression of c-Myc and cyclin D1. Furthermore, perivascular infiltration by inflammatory cells was elevated and inflammatory cells in BAL fluid were increased. Expression of Flk-1 (vascular endothelial growth factor receptor 2) and FoxF1, known regulators of pulmonary inflammation, was decreased in enFoxm1(-/-) mice. siRNA-mediated knockdown of FoxM1 in endothelial cells reduced Flk-1 and FoxF1 expression, which was driven by direct transcriptional induction by FoxM1 as target genes. Endothelial specific deletion of FoxM1 in vivo or in vitro also decreased expression of Sfrp1 (secreted frizzled-related protein 1), a known inhibitor of canonical Wnt signaling, in a manner that was associated with increased Wnt signaling. Taken together, our results suggest that endothelial-specific expression of FoxM1 limits lung inflammation and canonical Wnt signaling in lung epithelial cells, thereby restricting lung tumorigenesis.

Direct evidence of the vasodilator action of carbon dioxide on subcutaneous microvasculature in rats by use of intra-vital video-microscopy

The therapeutic effects of carbon dioxide (CO2) on cutaneous tissue blood flow in the human have long been well recognized. Although CO2 has vasodilator action, in-vivo evidence of its action on the microcirculation of the skin, and of its mechanism, has rarely been reported. We studied the direct effects of CO2 on in-vivo microvasculature and blood flow rate by using an intra-vital video-microscopic system. Brown Norway rats were anesthetized by intraperitoneal administration of alpha-chloralose and urethane. In order to measure inner diameter and red blood cell velocity (Vrbc) for a microvessel, the dorsal skin window was draped on an observation box placed inside a bath. Vrbc was derived from the cross-correlation function of paired segments of dual-window intensity in the video of microvascular images of the subcutaneous tissue. We measured pH in subcutaneous tissue by making a dorsal skin tube. After topical application of CO2 dissolved in water via the skin of the rat, we observed both vasodilatation and an increase in blood flow of the micro vessels. The pH of subcutaneous tissue also decreased after CO2 application. The CO2 reduced the pH of subcutaneous tissue and inhibited vascular smooth muscle contraction, resulting in dilatation of the vasculature of the skin microcirculation.

The effects of urethane on the isoflurane minimum alveolar concentration in rats

Urethane is often used as a sole anaesthetic agent for non-recovery studies in laboratory animals. However, the use of urethane is controversial, in part, because the electroencephalogram after urethane administration is similar to the electroencephalogram recorded from unanaesthetized animals. Here, we assessed the minimum alveolar concentration (MAC)-sparing effects of urethane by measuring the effect of two doses of urethane on the MAC of isoflurane in male Sprague Dawley rats. Isoflurane MAC was measured before and after intravenous administration of urethane at 1.0 g/kg (Group G₁, n = 6) and 1.5 g/kg (Group G₁.₅, n = 6), or an equal volume of 0.9% saline (Group Gs, n = 6). Baseline isoflurane MAC was not statistically different between groups (isoflurane concentration: 1.47 ± 0.08%, 1.40 ± 0.19% and 1.42 ± 0.12% for G₁, G₁.₅ and Gs, respectively). Intravenous injection of saline did not alter isoflurane MAC (post-saline MAC: 1.43 ± 0.11%). After urethane administration, isoflurane MAC decreased in a dose-dependent manner (new MAC G₁: 0.19 ± 0.06%; G₁.₅: 0.03 ± 0.01%; P < 0.05). The isoflurane MAC after 1.5 g/kg urethane was not significantly different from room air isoflurane concentrations (0.01 ± 0.01%), demonstrating a 100% MAC reduction at this dose. In conclusion, high-dose urethane (1.5 g/kg intravenously) was suitable as a sole anaesthetic agent to prevent gross purposeful movement during the conditions of the study, whereas low-dose urethane (1.0 g/kg intravenously) was not.

Vascular Endothelial Growth Factor Receptor 2–Targeted Chemoprevention of Murine Lung Tumors

No clinically effective chemoprevention for lung cancer has been found. Angiogenesis is an early feature of both adenocarcinoma and squamous cell lung cancer. We investigated the effects of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) inhibition on lung carcinogenesis in a murine model of adenocarcinoma. The VEGFR-2 tyrosine kinase inhibitor, vandetanib, was given to FVB/N mice in chow for 7 days at varying doses to show pharmacologic activity by inhibition of VEGF-mediated VEFGR-2 and ERK phosphorylation. Plasma levels corroborated adequate dosage. For chemoprevention experiments, mice were injected i.p. with 1 mg/g of urethane, a carcinogen found in tobacco smoke. Chow containing vandetanib, 75 mg/kg/d, or control chow was given to mice, starting 7 days after urethane administration. Sixteen weeks after urethane injection, mice were sacrificed, tumors enumerated and measured. Vandetanib resulted in reductions in tumor multiplicity (6.5 +/- 0.86 versus 1.0 +/- 0.30, P = 0.001) and average tumor volume (0.85 +/- 0.10 versus 0.15 +/- 0.09 mm(3), P = 0.001), but not incidence (71% versus 100%, P = ns), compared with control. As vandetanib has other activities besides VEGFR-2 tyrosine kinase inhibition, we gave the anti-VEGFR-2 monoclonal antibody, DC101, for weeks 11 to 15 of a urethane carcinogenesis protocol with an arrest in tumor volume increase, but no change in multiplicity or incidence. Further investigation of the chemopreventive effect of vandetanib and other VEGF signaling inhibitors is needed.

FTY720 and lung tumor development

FTY720 has been shown to prevent cancer development in experimental models but there is no report whether this beneficial effect is associated with the time point of the drug administration. Lung adenoma was induced in mice by urethane injection followed by different periods of FTY720 administration in order to evaluate lung tumor development. BALB/c mice received urethane intraperitoneally in two doses of 1.5 g/kg and were submitted to five daily doses of FTY720 (1 mg/kg/day) starting just after urethane injection (G2 n = 5), 4 weeks after urethane injection (G3 n = 10), 8 weeks after urethane injection (G4 n = 10) and no FTY720 administration (G1 n = 5). Twenty-four weeks after urethane administration mice were evaluated for the number of leukocyte in blood, lymphocytes in spleen, and lungs were evaluated for changes in histology, PCNA and VEGF expression. Lung nodules were present in higher numbers both in non treated (G1; 0.0–7.0) and FTY720 treated 8 weeks after urethane injection (G4; 0.0–6.0). G4 Group also presented the highest number of papillary nodules. G1 and G4 groups presented the lower number of splenocytes and neutrophils. In early time FTY720 treated mice (G2) we observed a slight decrease in PCNA staining and also the lower percentage of VEGF intense staining. Therefore, our data suggest that the benefits of FTY720 treatment are time-dependent and when administered in early periods after lung tumor induction this drug could impair cancer development.