Interim Sacrifices Research Articles

<i>Curcuma longa</i> renal historestorative effects on sildenafil induced nephrotoxicity among male albino rats

Background: Sildenafil is a phosphodiesterase type 5 inhibitor mostly used in management of erectile dysfunction and pulmonary hypertension. On the other hand, Curcuma longa is herbal plant that is mostly used in diets among the Asian and African population and commonly used in treatment of respiratory, renal and dermatological diseases.
 Methods: A post-test only true experimental study design was utilized with 25 Albino rats grouped as follows: negative control, positive control (sildenafil 1µg/g bwt), low dose Curcuma longa (38.75mg/kg), medium dose Curcuma longa (77.5mg/kg) and high dose Curcuma longa (155mg/kg) each having 5 rats. Induction of nephrotoxicity using sildenafil was done for 15 days with an interim sacrifice for negative and positive control groups done on day 15. Experimental groups were sacrificed on day 22 after receiving Curcuma longa at respective doses. Blood samples for renal function tests were obtained on day 1, 15 and 22. Enblock harvesting of the kidneys after euthanizing the rats with concentrated CO2 and 10% neutral buffered formalin perfusion was done on day 15 and 22 then fixed in 10% neutral buffered formalin for 24hours. The kidneys were then processed for Hematoxylin and Eosin staining and photomicrographs were taken using Olympus light microscope fitted with LABOMED ivu 3100 digital camera.
 Results: In sildenafil induced nephrotoxicity group; glomerulus was distorted, dilated bowman`s space, dilated renal tubules and necrotic epithelial cells. Minimal histological changes were observed in low Curcuma longa dose group while in medium and high Curcuma longa dose groups; the glomerulus had well defined margins, bowman`s space reduced in size, epithelial cells appeared normal.
 Conclusion: It can be concluded that medium and high dose Curcuma longa have kidney historestorative effects in sildenafil induced nephrotoxicity among male albino rats.

Safety Assessment of AAV2-hGDNF Administered Via Intracerebral Injection in Rats for Treatment of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neuroprotective biologic in Parkinson's disease models. Adeno-associated viral vector serotype 2 (AAV2)-human GDNF safety was assessed in rats treated with a single intracerebral dose of vehicle, 6.8 × 108, 6.8 × 109, or 5.2 × 1010 vector genomes (vg)/dose followed by interim sacrifices on day 7, 31, 90, and 376. There were no treatment-related effects observed on food consumption, body weight, hematology, clinical chemistry, coagulation parameters, neurobehavioral parameters, organ weights, or serum GDNF and anti-GDNF antibody levels. Increased serum anti-AAV2 neutralizing antibody titers were observed in the 5.2 × 1010 vg/dose group. Histopathological lesions were observed at the injection site in the 6.8 × 109 vg/dose (day 7) and 5.2 × 1010 vg/dose groups (days 7 and 31) and consisted of gliosis, mononuclear perivascular cuffing, intranuclear inclusion bodies, and/or apoptosis on day 7 and mononuclear perivascular cuffing on day 31. GDNF immunostaining was observed in the injection site in all dose groups through day 376 indicating no detectable impacts of anti-AAV2 neutralizing antibody. There was no evidence of increased expression of calcitonin gene-related peptide or Swann cell hyperplasia in the cervical and lumbar spinal cord or medulla oblongata at the 5.2 × 1010 vg/dose level indicating lack of hyperplastic effects. In conclusion, no systemic toxicity was observed, and the local toxicity observed at the injection site appeared to be reversible demonstrating a promising safety profile of intracerebral AAV2-GDNF delivery. Furthermore, an intracerebral dose of 6.8 × 108 AAV2-GDNF vg/dose was considered to be a no observed adverse effect level in rats.

Abstract 3966: The procancer effects of obesity can be reversed by moderate weight loss or an anti-inflammatory drug in a mouse model of colon cancer

Abstract Purpose: Obesity is associated with an increased risk of colon cancer. Our current study examines whether moderate weight loss or treatment with the non-steroidal anti-inflammatory drug sulindac suppresses the tumor-promoting effects of obesity in a mouse model of colon cancer. Methods: Male FVB mice were treated with azoxymethane (10 mg/kg i.p.) for 5 weeks, then randomized to a control (10% kcal from fat) or diet-induced obesity (DIO, 60% kcal from fat) diet. After 15 weeks, there was an interim sacrifice of 5 mice/group. The DIO mice were then further randomized to remain on DIO or switch to control diet to induce weight loss and become formerly obese mice (FOB), and within each diet group (control, DIO, and FOB), half the mice were randomized to start sulindac treatment (140 ppm in the diet). Eight weeks later, all mice were euthanized. Results: The DIO mice, compared to controls, had significantly greater body weight and body fat (P<0.05), while FOB mice had intermediate levels of adiposity. Sulindac did not affect adiposity in any diet group. At the interim timepoint, DIO mice had 10-fold greater tumor multiplicity relative to control mice (P=0.07). At study endpoint, DIO mice had greater tumor multiplicity compared to both control (P<0.05) and FOB (P<0.01) mice, which did not significantly differ from each other. Sulindac reduced tumor multiplicity in both control (P<0.05) and DIO (P<0.0001) mice, but not FOB mice. Serum levels of several inflammation-related proteins, including interleukin 6, chemokine (C-X-C motif) ligand 1, monocyte chemoattractant protein 1, and vascular endothelial growth factor, were elevated in DIO mice versus control (P<0.05) and DIO+sulindac (P<0.01) mice, but not in comparison to FOB mice. Gene expression in paired colon tumor and mesenteric adipose tissue samples was assessed by microarray. Ingenuity pathway analysis (IPA) of the significant (P<0.05) differentially expressed tumor genes indicated that both weight loss and sulindac reduced signaling related to matrix metalloproteinases (MMPs) and inflammation. In the visceral adipose tissue, IPA demonstrated that weight loss reduced signaling related to B cell receptors and glucose/fatty acid metabolism while sulindac decreased MMP, prostaglandin, and PPAR signaling. Additional analyses to explore the potential role of cross-talk between these two tissues are being performed, and key findings will be validated in the tissues at the protein level and via mechanistic cell culture experiments. Conclusions: Both moderate weight loss and sulindac treatment completely reverse the effects of chronic obesity on colon tumorigenesis, the latter independent of any change in adiposity. Our findings suggest that an investigation regarding the effects of NSAID treatment on colon cancer risk and/or progression in obese patients is warranted, particularly for those unable to achieve moderate weight loss. Citation Format: Laura W. Bowers, Arunima Punjala, Alison Q. Jeffries, Stephanie A. Montgomery, Andrew J. Dannenberg, Stephen D. Hursting. The procancer effects of obesity can be reversed by moderate weight loss or an anti-inflammatory drug in a mouse model of colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3966.

A Kinetic Analysis of DNA-Deoxy Guanine Adducts in the Nasal Epithelium Produced by Inhaled Formaldehyde in Rats-Assessing Contributions to Adduct Production From Both Endogenous and Exogenous Sources of Formaldehyde.

Although formaldehyde is a normal constituent of tissues, lifetime inhalation exposures at 6 h/day, 5 days/week at concentrations ≥6 ppm caused a nonlinear increase in nasal tumors in rats with incidence reaching close to 50% at 15 ppm. Studies with heavy isotope labeled [13CD2]-formaldehyde permit quantification of both the mass-labeled exogenous and endogenous DNA-formaldehyde reaction products. An existing pharmacokinetic model developed initially to describe 14C-DNA-protein crosslinks (DPX) provided a template for describing the time course of mass-labeled adducts. Published datasets included both DPX and N2-HO13CD2-dG adducts measured after a single 6-h exposure to 0.7, 2, 6, 9, 10, or 15 ppm formaldehyde, after multi-day exposures to 2 ppm for 6 h/day, 7 days/week with interim sacrifices up to 28 days, and after 28-day exposures for 6 h/day, 7 days/week to 0.3, 0.03, or 0.001 ppm. The existing kinetic model overpredicted endogenous adducts in the nasal epithelium after 1-day [13CD2]-formaldehyde exposure, requiring adjustment of parameters for rates of tissue metabolism and background formaldehyde. After refining tissue formaldehyde parameters, we fit the model to both forms of adducts by varying key parameters and optimizing against all 3 studies. Fitting to all these studies required 2 nonlinear pathways—one for high-exposure saturation of clearance in the nasal epithelial tissues and another for extracellular clearance that restricts uptake into the epithelial tissue for inhaled concentrations below 0.7 ppm. This refined pharmacokinetic model for endogenous and exogenous formaldehyde acetal adducts can assist in updating biologically based dose-response models for formaldehyde carcinogenicity.

Histopathology re-examination of the NTP toxicity/carcinogenicity studies of tert-butyl alcohol to identify renal tumor and toxicity modes of action

Tert-butyl alcohol (TBA) targets the rat kidney following repeated exposures, including renal tubule tumors. The mode of action (MOA) of these tumors, concluded by a pathology working group, involves both alpha2u-globulin nephropathy (α2u-gN) and exacerbated chronic progressive nephropathy (CPN), but has been disputed and an undefined MOA proposed. This study further reviews the histology slides of male and female rat kidneys from the NTP drinking water 13-week toxicity and 2-year carcinogenicity studies, including the 15-month interim sacrifice group. The papillary epithelial lining alteration formerly referred to as “transitional cell hyperplasia” develops as part of advanced CPN and does not represent a separate toxicity. No changes were observed in the kidney pelvis urothelium. The only alterations in subchronic male rats involved α2u-gN and CPN, without test article-related alterations in females. Focused examination of areas of parenchyma unaffected by CPN in TBA-treated male and female rats of the chronic studies revealed no renal tubule abnormalities other than from the effects of α2u-gN and CPN. Unrelated to toxicity were spontaneous amphophilic or vacuolar tubule proliferative lesions. All observed TBA-associated non-neoplastic and neoplastic histopathological changes in the kidney can be explained by α2u-gN or enhanced CPN, neither of which are relevant to humans.

A lifetime aging study of human CD19 transgenic mice.

Mice transgenic for human CD19 have been an important animal model to help understand the role of this molecule in B lymphocyte function. Previously, no lifetime studies had been performed to understand the effects of this CD19 over expression on the survival or spontaneous pathology within the C57BL/6J background strain. We conducted a lifetime study with interim sacrifices to understand the transgenic effects on clinical signs, body weight, survival, and spontaneous pathology. Blood and urine samples were collected from select animals at various time points during the study for measurement of clinical pathology parameters and groups of animals were euthanized and examined at predetermined intervals. There was fair survival with some animals living to 108weeks of age. Clinical pathology evaluations revealed a declining red cell mass with a regenerative anemia, increasing total white blood cell counts and decreasing glucose level. Total protein, albumin, and globulin levels increased to 52weeks of age and then declined to or below baseline with advancing age. Increased urinary microalbumin levels correlated with the severity of a glomerulopathy at 76 and 84weeks of age. Mean body weight increased through 70weeks and then declined to weights similar to week 28 at 108weeks. Macroscopic observations included pale kidneys, enlarged seminal vesicles, and enlarged spleens (at 108weeks of age). The most common neoplasms in this study were bronchiolar alveolar adenomas in the lung, histiocytic sarcoma in several different tissues, and hepatocellular adenomas. The most common non-neoplastic lesions were renal glomerulopathy, and pulmonary lymphocytic infiltrates with increased numbers of alveolar macrophages.

Toxicological Evaluation of β-Caryophyllene Oil: Subchronic Toxicity in Rats.

In a subchronic toxicity study, administration of β-caryophyllene (BCP) oil by oral gavage to Wistar rats at dosages of 0, 150, 450, or 700 mg/kg/d for 90 days, including a 21-day recovery period, did not produce any significant toxicologic manifestations. The study design also included a 28-day interim sacrifice in the control and high-dose groups. The BCP oil test article was well tolerated as evidenced by the absence of major treatment-related changes in the general condition and appearance of the rats, neurobehavioral end points, growth, feed and water intake, ophthalmoscopic examinations, routine hematology and clinical chemistry parameters, urinalysis, and necropsy findings. The no observed adverse effect level was the highest dosage level administered of 700 mg/kg body weight/d for both male and female rats. The study was conducted as part of an investigation to examine the safety of BCP oil for its proposed use in medical food products.

Safety and efficacy of a novel Prunus domestica extract (Sitoprin, CR002) on testosterone-induced benign prostatic hyperplasia (BPH) in male Wistar rats

The efficacy of a novel Prunus domestica bark extract (Sitoprin, CR002) was investigated on testosterone propionate (TP)-induced benign prostatic hyperplasia (BPH) in male Wistar rats. BPH was induced by daily subcutaneous administration of TP (3.0 mg/kg) over a period of 15 days (interim sacrifice group) and for an additional 21 days (terminal sacrifice group). We evaluated the dose-dependent efficacy (0, 50, 100 and 200 mg/kg body weight/day) of CR002 and a control group against BPH, and compared with a reference standard Prunus africana extract (CR001). Extensive clinical examinations were carried out on days 1, 7, 14, 21, 28 and 35 of treatment period to determine the onset, duration and severity of clinical signs. Clinical pathology, hematology, biochemistry and histopathology were performed on days 15 and 35, prior to necropsy. Animals were fasted overnight prior to blood collection. Prostate glands and tissues were examined. On day 36, histopathology of ventral prostrate of control rats demonstrates single layer of columnar mucin secreting epithelial cells along with a lumen occupied with eosinophilic secretion. In contrast, CR002 and CR001 groups (100 and 200 mg/kg/day) exhibited no hyperplasia and proliferation of epithelial cells. Prostate histopathology of these treated groups was comparable with control rats. The hyperplasia and hypertrophy of prostrate was reduced to single-layered cell indicating the efficacy of CR002 and CR001. Overall, results demonstrate that CR002 exhibits therapeutic efficacy/activity in TP-induced BPH in rats, which is comparable to CR001.

Efficacy of Novel Pygeum Extract (CR002) on Testosterone‐Induced Benign Prostatic Hyperplasia (BPH) in Male Wistar Rats

The efficacy of a novel Prunus domestica extract (pygeum extract, CR002) was investigated on testosterone propionate (TP)‐induced BPH in male Wistar rats. BPH was induced by subcutaneous administration of TP (3.0 g/kg) over a period of 15 days (interim sacrifice group) and for 21 days (terminal sacrifice group). We evaluated the dose‐dependent efficacy (0, 50, 100 and 200 mg/kg body weight/day) of novel Prunus domestica extract (CR002) and a control group against BPH, and compared with a reference standard Prunus africana extract (CR001). Extensive clinical examinations were carried out on days 1, 7, 14, 21, 28 and 35 of treatment period to determine the onset, duration and severity of clinical signs. Clinical pathology, hematology, biochemistry and histopathology were performed on days 15 and 35, prior to necropsy. Animals were fasted overnight prior to blood collection. Prostate glands and tissues were examined. On day 36, histopathology of ventral prostrate of control rats demonstrate single layer of columnar mucin secreting epithelial cells and lumen was occupied with eosinophilic secretion. In contrast, CR002 and CR001 groups (100 and 200 mg/kg/day) exhibited no hyperplasia and proliferation of epithelial cells. Prostate histopathology of these treated groups was comparable with control rats. The hyperplasia and hypertrophy of prostrate was reduced to single layered cell indicating the efficacy of CR002 and CR001. Overall, results demonstrate that CR002 exhibits therapeutic efficacy/activity in TP‐induced BPH in rats, which is comparable to CR001.

90-Day dietary toxicity study with esterified propoxylated glycerol (EPG) in rats

The subchronic (90-day) toxicity of a “core” version of EPG was assessed in rats. Crl:CD-1®(ICR)BR rats (70/sex) received diets containing a constant level of 5% EPG (w/w) or adjusted to deliver 0 (control), 0.5, 1, or 2g/kg of body weight/day (g/kg bw/day). Subsets of animals from each group (20/sex) were evaluated after 30days (interim sacrifice); the remainder after 90days. EPG intake at all dose levels was associated with lower mean liver vitamin E levels; liver vitamin A and serum vitamin D were also lower, but less consistently. Animals given 5% EPG had higher fecal output (males) and cholesterol (males and females) without corresponding changes in serum cholesterol. Urinary pH was also mildly lower in males given 5% EPG. However, detailed evaluation of general health and assessment of blood, organs and tissues showed no evidence that EPG administration compromised the nutritional requirements of the animals, caused a state of fat-soluble vitamin deficiency, or caused‘ toxicity to any organ system. Based on the results of this study, it was not possible to establish a no-observable-effect level (NOEL). The possible effect of EPG on vitamin levels in the absence of any clinical signs of deficiency was not considered “adverse” per se. As such, the 2g/kg and 5% EPG level were considered to represent a no-observable-adverse-effect levels (NOAELs).

An autologously generated platelet-rich plasma suturable membrane may enhance peripheral nerve regeneration after neurorraphy in an acute injury model of sciatic nerve neurotmesis.

The aim of this study was to investigate the ability of suturable platelet-rich plasma (PRP) membrane to promote peripheral nerve regeneration after neurotmesis and neurorraphy. A total of 36 rats were used: 32 animals underwent surgery and were split in two groups. An interim sacrifice was performed at 6 weeks postsurgery and final sacrifice at 12 weeks; four animals did not sustain nerve injury and served as control. Clinical, electromyographic (EMG), gross, and histological changes were assessed. The EMG signal was evaluated for its amplitude and frequency spectrum. Number of regenerating fibers, their diameter, and myelin thickness were histologically analyzed. Both EMG parameters showed a significant (p < 0.05) effect of treatment at 6 and 12 weeks postsurgery. At 6 weeks, the fiber density was statistically different between treated and untreated animals with a higher observed density in treated nerves. No difference in fiber density was observed at 12 weeks postsurgery. The distribution of fiber diameters showed an effect at 12 weeks when only the sections of the nerves sutured with PRP showed fibers with diameters greater than 6 µm. Our data show that the application of a PRP fibrin membrane around the neurorraphy improves the nerve regeneration process in a rat sciatic nerve model. The use of PRP as a suturable membrane could perform an action not only as a source of bioactive proteins but also as a nerve guide to hold the scar reaction and thus improve axonal regeneration.

Intestinal Lymphangiectasis and Lipidosis in Rats Following Subchronic Exposure to Indole-3-Carbinol via Oral Gavage

To investigate the toxicity and carcinogenic potential of indole-3-carbinol (I3C), the National Toxicology Program has conducted 13-week subchronic studies in Fisher 344 rats and B6C3F1 mice, and chronic 2-year bioassays in Sprague-Dawley rats and B6C3F1 mice. While the chronic study results are not yet available, subchronic study results and short-term special evaluations of interim sacrifices in the 2-year rat bioassay are presented. F344 rats were orally gavaged ≤300 mg I3C/kg body weight 5 days a week for 13 weeks. Rats treated with ≥150 mg/kg demonstrated a dose-related dilation of lymphatics (lymphangiectasis) of the duodenum, jejunum, and mesenteric lymph nodes. Material within dilated lacteals stained positively for Oil Red O and Sudan Black, consistent with lipid. Electron microscopic evaluation confirmed extracellular lipid accumulation within the villar lamina propria, lacteals, and within villar macrophages. Analyses of hepatic and pulmonary CYP1A enzymes demonstrated dose-dependent I3C induction of CYP1A1 and 1A2. B6C3F1 mice orally gavaged ≤250 mg I3C/kg body weight did not demonstrate histopathological changes; however, hepatic CYP induction was similar to that in rats. The histopathologic changes of intestinal lymphangiectasis and lipidosis in this study share similarities with intestinal lymphangiectasia as observed in humans and dogs. However, the resultant clinical spectrum of protein-losing enteropathy was not present.

Chronic dietary toxicity and carcinogenicity study with potassium perfluorooctanesulfonate in Sprague Dawley rats

To investigate toxicity and neoplastic potential from chronic exposure to perfluorooctanesulfonate (PFOS), a two-year toxicity and cancer bioassay was conducted with potassium PFOS (K+PFOS) in male and female Sprague Dawley rats via dietary exposure at nominal K+PFOS concentrations of 0, 0.5, 2, 5, and 20μg/g (ppm) diet for up to 104 weeks. Additional groups were fed 20ppm for the first 52 weeks, after which they were fed control diet through study termination (20ppm Recovery groups). Scheduled interim sacrifices occurred on Weeks 4, 14, and 53, with terminal sacrifice between Weeks 103 and 106. K+PFOS appeared to be well-tolerated, with some reductions in body weight occurring in treated rats relative to controls over certain study periods. Male rats experienced a statistically significant decreased trend in mortality with significantly increased survival to term at the two highest treatment levels. Decreased serum total cholesterol, especially in males, and increased serum urea nitrogen were consistent clinical chemistry observations that were clearly related to treatment. The principal non-neoplastic effect associated with K+PFOS exposure was in livers of males and females and included hepatocellular hypertrophy, with proliferation of endoplasmic reticulum, vacuolation, and increased eosinophilic granulation of the cytoplasm. Statistically significant increases in hepatocellular adenoma were observed in males (p=0.046) and females (p=0.039) of the 20ppm treatment group, and all of these tumors were observed in rats surviving to terminal sacrifice. The only hepatocellular carcinoma observed was in a 20ppm dose group female. There were no treatment-related findings for thyroid tissue in rats fed K+PFOS through study termination; however, male rats in the 20ppm Recovery group had statistically significantly increased thyroid follicular cell adenoma, which was considered spurious. There was no evidence of kidney or bladder effects. In rats, the dietary dose estimated as the lower 95% confidence limit of the benchmark dose for a 10% increase in hepatic tumors was 8ppm for both sexes. Recent mechanistic studies suggest a PPARα/CAR/PXR-mediated mode of action for the liver tumors observed in the present two-year study.

Preclinical Safety Evaluation of AAV2-sFLT01— A Gene Therapy for Age-related Macular Degeneration

AAV2-sFLT01 is a vector that expresses a modified soluble Flt1 receptor designed to neutralize the proangiogenic activities of vascular endothelial growth factor (VEGF) for treatment of age-related macular degeneration (AMD) via an intravitreal injection. Owing to minimal data available for the intravitreal route of administration for adeno-associated virus (AAV), we initiated a 12-month safety study of AAV2-sFLT01 administered intravitreally at doses of 2.4 × 10(9) vector genomes (vg) and 2.4 × 10(10) vg to cynomolgus monkeys. Expression of sFlt01 protein peaked at ~1-month postadministration and remained relatively constant for the remainder of the study. Electroretinograms, fluorescein angiograms, and tonometry were assessed every 3 months, with no test article-related findings observed in any group. Indirect ophthalmoscopy and slit lamp exams performed monthly revealed a mild to moderate but self-resolving vitreal inflammation in the high-dose group only, which follow-up studies suggest was directed against the AAV2 capsid. Histological evaluation revealed no structural changes in any part of the eye and occasional inflammatory cells in the trabecular meshwork, vitreous and retina in the high-dose group. Biodistribution analysis in rats and monkeys found only trace amounts of vector outside the injected eye. In summary, these studies found AAV2-sFLT01 to be well-tolerated, localized, and capable of long-term expression.

Thyroid Follicular Lesions Induced by Oral Treatment for 2 Years with 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Dioxin-like Compounds in Female Harlan Sprague-Dawley Rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and structurally-similar dioxin-like compounds affect thyroid function and morphology and thyroid hormone metabolism in animals and humans. The National Toxicology Program conducted eight 2-year gavage studies in female Harlan Sprague-Dawley rats to determine the relative potency of chronic toxicity and carcinogenicity of TCDD, 3,3',4,4',5-pentachlorobiphenyl (PCB126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 2,3',4,4',5-pentachlorobiphenyl (PCB118), 2,2',4,4',5,5'-hexachloro-biphenyl (PCB153), a tertiary mixture of TCDD/PCB126/PeCDF, and two binary mixtures (PCB126/PCB153 and PCB126/PCB118). Administration of these compounds was associated with increased incidences of thyroid follicular cell hypertrophy, variably observed in the 14-, 31-, and 53-week interim and 2-year sacrifice groups. In all studies, the incidences of follicular cell adenoma and carcinoma were not increased. Decreased levels of serum thyroxine were primarily noted in the 14-or-later -week interim groups of all chemicals. Serum triiodothyronine (T3) levels were increased in the TCDD, PCB126, PeCDF, TCDD/PCB126/PeCDF, and PCB126/PCB153 studies, while decreased levels were noted in the PCB153 and PCB126/PCB118 studies. TCDD, PCB126, PCB126/PCB153, and PCB126/PCB118 increased levels of serum thyroid-stimulating hormone almost in a dose-dependent manner in the 14-week groups. These data suggest that although dioxin-like compounds alter thyroid hormones and increase follicular cell hyperplasia, there is not an increase in thyroid adenoma or carcinoma in female Sprague-Dawley rats.

Pathogenicity of the entomopathogenic fungus Beauveria bassiana 9205 in rats using different routes of exposition

Among fungi intended for biological control, great efforts have been made in the development of entomopathogenic Hyphomycetes, like Beauveria bassiana, which have a wide host range. The aim of this study was to assess and to compare the pathogenicity of B. bassiana strain 9205 in rats through oral, intranasal, and intravenous dosing. Through 21 days after administration, clinical examinations were performed daily, and body weight gain was evaluated. Clearance was estimated by means of collection of feces or examination of lungs and blood, and infectivity was evaluated enumerating microorganisms from organs of animals sacrificed at intervals. Gross necropsy of animals was performed at interim or final sacrifice. There were no mortalities, and no evidence of pathogenicity or treatment-related toxicity, although the microorganism was capable of achieving significative infectivity in organs after intravenous administration. The obtained results suggest the innocuity of B. bassiana in rats and the necessity of more prolonged studies through intravenous injection.

Estimation of Lag Time Between Onset of and Death from an Occult Tumor

A new statistical method for estimating the lag time between onset of and death from an occult tumor is proposed for data without cause-of-death information. In this method, the survival function for time to tumor onset, tumor-specific survival function, and competing risks survival function are estimated using the maximum likelihood estimates of the parameters. The proposed method utilizes the estimated survival functions and statistically imputed fatal tumors to estimate the lag time. This approach is developed for rodent tumorigenicity assays that have at least one interim sacrifice and a terminal sacrifice. If the data contain cause-of-death information given by pathologists and it is believed to be reliable, it may be used for estimating the lag time. The proposed method is illustrated using a real data set. The accuracy of the estimation of lag time is evaluated via a Monte Carlo simulation study. This study shows that the estimated lag time is quite reliable.

Imaging Update: New Windows, New Views

The recent progress in our understanding of the molecular-genetic mechanisms active in many diseases and the application of new biologically based approaches in therapy are exciting new developments that have emerged following mapping of the human genome. Novel “molecular therapies” have been

Chronic Preclinical Safety Evaluation of Hematide™, a PEGylated Peptidic Erythropoiesis Stimulating Agent, in Monkeys.

Hematide, a synthetic PEGylated peptidic compound specifically binds and activates the erythropoietin receptor thereby stimulating erythropoiesis. To support chronic clinical dosing requirements, a comprehensive safety program was undertaken including a 9-month IV Cynomolgus monkey toxicology study. Four groups, composed of 16 male and 16 females in the vehicle-control and high dose groups and 8 male and 8 females in the low and mid-dose groups, received 0, 0.2, 2, and 20 mg/kg Hematide IV Q3W for up to 9 months followed by a 14-week recovery. An interim sacrifice was conducted on days 90 and 195. Clinical observations including ophthalmologic examinations, body weight, and food consumption were recorded. Clinical pathologic evaluations, organ weight, histopathologic, and ECG examinations were performed. Toxicokinetics and anti-Hematide antibodies were also assessed. Unscheduled deaths included 3 high-dose animals on Days 79, 104 and 207. Chronic administration was associated with polycythemia that was time and dose-dependent. There were no gender-related differences. At Day 90, the average Hgb levels for males were 13.9, 17.0, 21.7, and 23.3 g/dL for 0, 0.2, 2.0, and 20 mg/kg, respectively, corresponding to Hgb increases, over controls, of 3.1, 7.8, and 9.4 g/dL. Thereafter, Hgb values maintained peak levels. At Day 279, for the males, the average Hgb levels were 13.7, 15.5, 21.9, and 23.0 g/dL for 0, 0.2, 2.0, and 20 mg Hematide/kg, respectively, corresponding to Hgb increases, over controls, of 1.8, 8.2, and 9.3 g/dL. During the recovery period, hematology values steadily decreased to values more similar to controls. By Day 372, approximately fourteen weeks after the final dose of Hematide, for males, the average Hgb levels were 12.7, and 11.7 g/dL for 0 and 20 mg/kg, respectively. The findings in the three high dose animals that died prior to sacrifice indicated that the cause of death was secondary to hemodynamic changes that were attributed to exaggerated polycythemia induced by very high and repeated Hematide doses. Effects on the micro-vasculature of several organ sections (brain, kidney, liver, lung, spleen, and gastro-intestinal tract) were observed for monkeys dosed at ≥ 2.0 mg/kg. The return to normalcy from suspected test article related lesions was generally complete in monkeys euthanized after a 14-week recovery period for the 20.0 mg/kg group except for effects on choroid plexus and kidneys which were still detectable. Hematide displayed a sustained plasma level following intravenous administration in monkeys. At all dose levels and dosing intervals, increases in AUC values were greater than dose proportional, indicating a deviation from linear kinetics over the dose range studied. Apparent half-life (t1/2) at steady state (Day 106) ranged from ~30 h at the low dose to ~90 h at the high dose. Three of 32 monkeys in the 20 mg/kg group were deemed likely positive for anti-Hematide antibodies but the antibodies were not cross-reactive with erythropoietin. No antibodies were detected in the low and medium dose groups. No difference in pharmacology and toxicology profiles were observed in the 3 antibody-positive monkeys compared to antibody-negative monkeys. In conclusion, Hematide is a potent ESA with prolonged half-life and this study supports chronic clinical administration for the correction of anemia associated with chronic kidney disease and cancer.

Carbohydrate Digestibility Predicts Colon Carcinogenesis in Azoxymethane-Treated Rats

: The purpose of this study was to compare the effect of carbohydrate structure and digestibility on azoxymethane (AOM)-induced colon carcinogenesis. Five groups of male Fischer 344 rats each comprising 30 animals were injected with AOM and fed a high-fat diet with 15% of various carbohydrates. The carbohydrate sources used were sucrose, cornstarch (a linear starch, reference group), potato starch (a branched starch), a short-chained oligofructose (Raftilose®), and a long-chained inulin-type fructan (Raftiline®). An interim sacrifice was performed after 9 wk to investigate markers of carbohydrate digestibility, including caecal fermentation (caecum weight and pH) and glucose and lipid metabolism [glucose, fructoseamine, HbA1c, triglycerides, and insulin-like growth factor (IGF) 1]. In addition potential early predictors of carcinogenicity [cell proliferation and aberrant crypt foci (ACF)] at 9 wk and their correlation to colon cancer risk after 32 wk were investigated. Tumor incidence was significantly reduced in animals fed oligofructose, and the number of tumors per animal was significantly reduced in animals fed inulin and oligofructose at 32 wk after AOM induction compared to the reference group fed sucrose. Increased caecum weight and decreased caecal pH were seen in groups fed oligofructose, inulin, and potato starch. Plasma triglyceride was decreased in rats fed oligofructose and inulin. Cell proliferation was increased in the proximal colon of rats fed sucrose, oligofructose, and inulin, and the number of cells per crypt decreased in rats fed oligofructose and inulin. The total number of ACF's was unaffected by treatment, and the size and multiplicity of ACF was unrelated to tumor development. It was concluded that less digestible carbohydrates with an early effect on caecum fermentation and plasma triglyceride decreased subsequent tumor incidence and multiplicity. This was unrelated to ACF, cell proliferation, and other markers of glucose and lipid metabolism.