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PubMed1999

Diet, caloric restriction, and the rodent bioassay.

The diet can significantly alter the results of toxicity and carcinogenicity studies. Ad libitum (AL) overfeeding of excessive calories to sedentary adult rodents is one of the most poorly controlled variables affecting the current rodent bioassay. AL-overfed rodents develop an early onset of adverse metabolic events, endocrine-disruptive degenerative diseases, and tumors that result in early morbidity and mortality. AL food consumption is extremely variable, but has a strong correlation with adult body weight, obesity, and survival. AL feeding of diets with modified protein, fiber, and energy content are not as effective as simple, moderate dietary (caloric) restriction (DR) in controlling these study variables. Moderate DR (70-75% of adult AL) is operationally simple and controls adult body weights, prevents obesity, and improves health and survival by reducing or delaying diet-related endocrine, renal, and cardiac diseases. Moderate DR provides a uniform rodent model, increases treatment exposure time, and increases the statistical sensitivity of these chronic bioassays to detect true treatment effects. Feeding a balanced diet by a moderate DR regimen of 70-75% of the maximum, unrestricted adult AL food intake is recommended for conducting well-controlled toxicity and carcinogenicity studies.

Animals↗
PubMed2006

Mammalian monogamy is not controlled by a single gene.

Complex social behavior in Microtus voles and other mammals has been postulated to be under the direct genetic control of a single locus: the arginine vasopressin 1a receptor (avpr1a) gene. Using a phylogenetic approach, we show that a repetitive element in the promoter region of avpr1a, which reportedly causes social monogamy, is actually widespread in nonmonogamous Microtus and other rodents. There was no evidence for intraspecific polymorphism in regard to the presence or absence of the repetitive element. Among 25 rodent species studied, the element was absent in only two closely related nonmonogamous species, indicating that this absence is certainly the result of an evolutionarily recent loss. Our analyses further demonstrate that the repetitive structures upstream of the avpr1a gene in humans and primates, which have been associated with social bonding, are evolutionarily distinct from those in rodents. Our evolutionary approach reveals that monogamy in rodents is not controlled by a single polymorphism in the promoter region of the avpr1a gene. We thus resolve the contradiction between the claims for an evolutionarily conserved genetic programming of social behavior in mammals and the vast evidence for highly complex and flexible mating systems.

Animals↗
PubMed2005

Rodent StAR mRNA is substantially regulated by control of mRNA stability through sites in the 3'-untranslated region and through coupling to ongoing transcription.

The steroidogenic acute regulator (StAR) gene is transcribed to 1.6 kb and 3.5 kb mRNAs that differ only through the length of the 3'-untranslated region (3'-UTR). These forms result from alternative polyadenylation sites in exon 7. These sites are utilized similarly in unstimulated adrenal cells whereas Br-cAMP selectively stimulates 3.5 kb mRNA. After removal of Br-cAMP, 3.5 kb mRNA declines rapidly (t(1/2) = 2 h) while 1.6 kb mRNA responds more slowly. This selective degradation is more evident in testis MA10 cells and is seen even in the presence of Br-cAMP. Transfection of Y-1 cells with CMV promoted StAR vectors confirmed that the 3.5 kb form is less stable and that Br-cAMP slowly increases this instability. Basal instability resides solely in the extended 3'-UTR which contains AU-rich elements. Br-cAMP enhances this degradation of 3.5 kb mRNA but additionally requires translated and 5'-UTR sequences. Degradation of both forms is arrested by inhibitors of transcription or translation, indicating that mRNA stability is coupled to these processes independent of the extended 3'-UTR. Br-cAMP stimulates substantial selective synthesis of 3.5 kb StAR mRNA despite this instability. The preferential generation of the unstable form may facilitate rapid increases and decreases of StAR activity in response to external changes.

3' Untranslated Regions↗
PubMedDate not supplied

Reduction of intestinal neoplasia with adenomatous polyposis coli gene replacement and COX-2 inhibition is additive.

Mutations of the adenomatous polyposis coli (APC) gene are implicated early in colorectal tumorigenesis. Restoration of normal APC expression through gene therapy may prevent or reduce intestinal neoplasia. Furthermore, the relationship between colorectal tumors and increased cyclooxygenase-2 (COX-2) activity provides a rationale for the use of selective COX-2 inhibitors such as rofecoxib (Vioxx) to prevent the formation of polyps. This study was performed to determine the effects of liposome-mediated APC gene therapy and a selective COX-2 inhibitor on intestinal neoplasia in vivo. Five-week-old Min mice weaned on a 30% high-fat diet were randomized to receive no treatment (control), APC only, Vioxx only, and APC/Vioxx. APC-treated mice received a plasmid containing the human APC cDNA (pCMV-APC) mixed with a liposome preparation that was administered biweekly. Vioxx was administered at 200 ppm in the high-fat rodent chow. The control mice were treated similarly with a plasmid construct lacking the APC gene. Confirmation of exogenous APC gene expression was determined by Western blot analysis. After 2 months, there was a 54% and 70% reduction in the total number of intestinal polyps after APC and Vioxx treatment, respectively. Combined APC/Vioxx therapy reduced polyp formation by 87%. The reduction of intestinal neoplasia by APC gene replacement and COX-2 inhibition suggests their separate roles in intestinal tumorigenesis. Each modality, both individually and together, may prove therapeutic and therefore contribute to new strategies in the prevention and treatment of colorectal cancer.

Animals↗
PubMed1998

The effects of twisted ischaemic adnexa managed by detorsion on ovarian viability and histology: an ischaemia-reperfusion rodent model.

This prospective controlled follow-up study was designed to examine the effects of adnexal torsion on long-term ovarian histology and radical scavenger (FRS) activity, and subsequent viability following the detorsion of twisted ischaemic adnexa, in a primate centre of a university clinic. Adnexal torsion/occlusion was created by twisting the adnexa three times and fixing on to the side wall or by applying vascular clips in cycling female rats at 70 days of age. Following an ischaemic period of 4 to 36 h, the twisted adnexas were surgically removed and fixed. In the second group of rats, following the above ischaemic periods, the torsion/occlusion were relieved by detwisting or removing the vascular clips. Then the animals were reperfused for a week and adnexas were extirpated. After both ischaemia and reperfusion, the removed adnexas were examined histologically and tissue concentrations of glutathione peroxidase, superoxide dismutase, catalase and glutathione were determined. Regardless of the ischaemia time, all the twisted adnexas were black-bluish in appearance. Despite the gross ischaemic-haemorrhagic features, histological sections revealed negligible changes, with intact ovarian structure similar to controls in 4-24 h groups. Though decreased compared with controls, the change in tissue concentrations of FRS was not significant in 4-24 h groups. Only the 36 h group showed prominent congestion on all sections and a significant decrease in all radical scavenger concentrations studied. While no long-term reperfusion injury was observed histologically in 4-24 h groups, the 36 h group ended with adnexal necrosis. Our findings support the importance of early diagnosis and conservative surgical management (detorsion) in adnexal torsion. Lack of histological changes and unimpaired FRS metabolism are consistent with the recent data that vascular compromise is caused by venous or lymphatic stasis in early torsion and that adnexal integrity is not correlated with gross ischaemic appearance, thus providing evidence of adnexal resistance against ischaemia.

Adnexa Uteri↗
PubMed2004

Rat endometrial Vdup1 expression: changes related to sensitization for the decidual cell reaction and hormonal control.

During implantation in rodents, attachment and invasion of embryonic trophoblast is accompanied by decidualization of the adjacent endometrial stroma. Decidualization can be initiated only when the endometrium is receptive, and this occurs for a short period in pregnancy. The molecular mechanisms underlying this phenomenon remain unclear. In the current study, using differential display and northern blot analysis, we found that steady-state levels of mRNA for vitamin D3 upregulated protein 1 (Vdup1) were significantly higher in 'refractory' and 'delayed' endometrium compared with 'receptive' endometrium or endometrium undergoing artificially induced decidualization. Conversely, thioredoxin (Txn), a ubiquitously expressed cellular redox regulator known to promote growth and proliferation, was found to have elevated transcript levels within the decidualizing endometrium. VDUP1 has previously been shown to bind TXN and inhibit its action. In an inverse, but cooperative, relationship, these molecules have been implicated in regulating cell growth and proliferation in a number of tissues and during transformation to cancer. The Vdup1 mRNA is localized to the uterine stroma in the nonreceptive endometrium, the site of increased Txn mRNA levels during decidualization. In addition, Vdup1 mRNA levels are inversely regulated by progesterone and estrogen; prolonged progesterone exposure stimulates an increase in Vdup1 mRNA levels whereas estrogen decreases Vdup1 transcript levels. Together, these results suggest a novel mechanism by which suppression of the decidual response in the nonreceptive endometrium may occur.

Animals↗
PubMed2004

In vitro biotransformation and genotoxicity of the drinking water disinfection byproduct bromodichloromethane: DNA binding mediated by glutathione transferase theta 1-1.

The drinking water disinfection byproduct bromodichloromethane (CHBrCl(2)) was previously shown to be mutagenic in Salmonella typhimurium that overexpress rat glutathione transferase theta 1-1 (GSTT1-1). Several experimental approaches were undertaken in this study to investigate the DNA covalent binding potential of reactive intermediates generated by GSTT1-1-mediated metabolism of CHBrCl(2). First, rodent hepatic cytosol incubations containing [(14)C]CHBrCl(2), supplemented glutathione (GSH), and calf thymus DNA resulted in approximately 3-fold (rat liver cytosol) and 7-fold (mouse liver cytosol) greater amounts of total radioactivity (RAD) associated with the purified DNA as compared to a control (absence of rodent cytosol) following liquid scintillation counting (LSC) of isolated DNA. The relative increase in DNA labeling is consistent with the conjugation activity of these rodent cytosols toward CHBrCl(2). Second, exposure of GSTT1-1-expressing S. typhimurium to [(14)C]CHBrCl(2) resulted in a concentration-dependent increase of bacterial DNA-associated total radioactivity. Characterization of DNA-associated radioactivity could not be assigned to a specific deoxynucleoside adduct(s) following enzymatic hydrolysis of DNA and subsequent HPLC analysis. A possible explanation for this observation was formation of a 'transient' adduct that was unstable in the DNA isolation and hydrolysis procedures employed. To circumvent problems of adduct instability, reactions of [(14)C]CHBrCl(2) with GSH catalyzed by recombinant rat GSTT1-1 were performed in the presence of calf thymus DNA or, alternatively, the model nucleophile deoxyguanosine. Hydroxyapatite chromatography of [(14)C]-labeled DNA or HPLC chromatography of [(14)C]-labeled deoxyguanosine derivatives demonstrated the covalent binding of [(14)C]CHBrCl(2)-derived metabolites to DNA and deoxyguanosine in low yield (approximately 0.02% of [(14)C]CHBrCl(2) biotransformed by GSTT1-1 resulted in DNA adducts). Cytochrome P450 (CYP)- and GST-catalyzed biotransformation of CHBrCl(2) in rat tissues (kidney and large intestine) that develop tumors following chronic CHBrCl(2) exposure were compared with rat liver (a nontarget tissue). Rat liver had a significant capacity to detoxify CHBrCl(2) (to carbon dioxide) compared with kidney and large intestine as a result of CYP-catalyzed oxidation, liver was approximately 16-fold more efficient than kidney and large intestine when intrinsic clearance values (V(max)/K(m)) were compared. In contrast, the efficiency of GST-mediated GSH conjugation of CHBrCl(2) in kidney and large intestine was only slightly lower than liver (approximately 2- to 4-fold lower), thus, the relative amounts of reactive intermediates that are produced with the capacity to covalently modify DNA may be enhanced in these extrahepatic tissues. The significance of these findings is that conjugation of CHBrCl(2) with GSH can result in the covalent modification of DNA and that cancer target tissues in rats have a much reduced detoxification capacity, but only a modest decrease in bioactivation capacity, as compared to the liver (a nontarget tissue in rats).

Animals↗
PubMed2006

A pressure-controlled rat ventilator with electronically preset respirations.

Major experimental surgery on laboratory animals requires adequate anesthesia and ventilation to keep the animal alive throughout the procedure. A ventilator is a machine that helps the anesthesized animal breathe through an endotracheal tube by pumping a volume of gas (oxygen, air, or other gaseous mixtures), comparable with the normal tidal volume, into the animal's lungs. There are two main categories of ventilators for small laboratory rodents: volume-controlled and pressure-controlled ones. The volume-controlled ventilator injects a preset volume into the animal's lungs, no matter the airways' resistance (with the peak inspiratory pressure allowed to vary), while the pressure ventilator controls the inspiratory pressure and allows the inspiratory volume to vary. Here we show a rat pressure ventilator with a simple expiratory valve that allows gas delivery through electronic expiration control and offers easy pressure monitoring and frequency change during ventilation.

Animals↗
PubMed2000

Dehydroepiandrosterone sulfate and beta-cell function: enhanced glucose-induced insulin secretion and altered gene expression in rodent pancreatic beta-cells.

Administration of dehydroepiandrosterone (DHEA), or its sulfated form (DHEAS), controls hyperglycemia in diabetic rodents without directly altering insulin sensitivity. We show that DHEAS enhanced glucose-stimulated insulin secretion when administered in vivo to rats or in vitro to beta-cell lines, without changing cellular insulin content. Insulin secretion increased from 3 days of steroid exposure in vitro, suggesting that DHEAS did not directly activate the secretory processes. DHEAS selectively increased the beta-cell mRNA expression of acyl CoA synthetase-2 and peroxisomal acyl CoA oxidase in a time-dependent manner. Although DHEAS is a peroxisomal proliferator, it did not alter the mRNA expression of peroxisomal proliferator-activated receptor (PPAR) alpha or beta, or enhance the activity of transfected PPAR alpha, beta, or gamma in vitro. Thus, DHEAS directly affected the beta-cell to enhance glucose-stimulated insulin secretion and increased the mRNA expression of specific beta-cell mitochondrial and peroxisomal lipid metabolic enzymes. This effect of DHEAS on insulin secretion may contribute to the amelioration of hyperglycemia seen in various rodent models of diabetes.

Acyl-CoA Oxidase↗
PubMed1989

Protection of rhesus monkeys from fatal Lassa fever by vaccination with a recombinant vaccinia virus containing the Lassa virus glycoprotein gene.

Lassa fever is an acute febrile disease of West Africa, where there are as many as 300,000 infections a year and an estimated 3000 deaths. As control of the rodent host is impracticable at present, the best immediate prospect is vaccination. We tested as potential vaccines in rhesus monkeys a closely related virus, Mopeia virus (two monkeys), and a recombinant vaccinia virus containing the Lassa virus glycoprotein gene, V-LSGPC (four monkeys). Two monkeys vaccinated with the New York Board of Health strain of vaccinia virus as controls died after challenge with Lassa virus. The two monkeys vaccinated with Mopeia virus developed antibodies measurable by radioimmunoprecipitation prior to challenge, and they survived challenge by Lassa virus with minimal physical or physiologic disturbances. However, both showed a transient, low-titer Lassa viremia. Two of the four animals vaccinated with V-LSGPC had antibodies to both Lassa glycoproteins, as determined by radioimmunoprecipitation. All four animals survived a challenge of Lassa virus but experienced a transient febrile illness and moderate physiologic changes following challenge. Virus was recoverable from each of these animals, but at low titer and only during a brief period, as observed for the Mopeia-protected animals. We conclude that V-LSGPC can protect rhesus monkeys against death from Lassa fever.

Animals↗
PubMed2006

Toxoplasmosis in piglets.

Seventeen-day-old piglets in a small holding farm in southern Thailand manifested signs of convulsion, fever, and death. The morbidity and mortality rate were approximately 26.09% (6/23) and 4.35% (1/23), respectively. Impression smear from lungs demonstrated tachyzoite stage of Toxoplasma gondii. Histopathological investigation revealed interstitial pneumonia. Further investigation, blood collection, educating the farmer, and prescribed affected herd with sulfa-trimethoprim were performed soon after Toxoplasma infection was demonstrated. A serological detection of T. gondii infection among affected herds using latex agglutination test was conducted on 14 serum samples. The overall seroprevalence was 71.43% (10/14). The titers were 1:64 in 3 pigs (21.43%), 1:128 in 4 (28.57%), 1:256 in 2 (14.29%), and 1:512 in 1 (7.14%). A cat in this farm showed antibody titer 1:32 while a dog was seronegative. Data derived from this case indicated two possible routes of transmission: transplacental infection and ingested food or water contaminated with oocysts shed by cat in this farm. However, rodents can not exclude as a role of toxoplasmosis transmission. Serological monitoring at slaughter houses combine with good sanitary practices, rodent and cat control in the farm are important measures to prevent toxoplasma infection and improve human health in the future.

Animal Feed↗
PubMed1990

Prolonged survival of female AKR mice fed diets supplemented with methionine and choline.

Female mice of the AKR/J (AK) strain were fed a control diet (Purina Rodent Laboratory Chow) or a lipotrope-supplemented diet (Purina Rodent Chow plus 2% D,L-methionine and 1% choline chloride) beginning at 1 day after weaning. Food consumption and weight gain were found to be the same in both groups of animals. Mice of this inbred strain spontaneously develop thymic lymphoma, with close to 100% mortality expected by 12-13 months of age. Two separate experiments were carried out with 50 mice per group in one, and 40 mice per group in the other. The slopes of the survival curves for the animals in the control group and supplemented group of mice diverged after the animals reached 6.5 months of age. In both experiments, 20% of the mice receiving supplemented diet were still alive at 1 year, while 3% in one experiment and 8% in the other experiment survived in the control groups. Each experiment was terminated when the animals reached 13 months of age. At that time the survival rate of the controls was 2 and 4%, and survival in the groups of mice receiving supplemented diet was 14 and 18%. Necropsy revealed that the animals in both groups had advanced malignant lymphoma. Our results demonstrate that intake of a chow diet that is supplemented with moderate quantities of methionine and choline results in enhanced survival of spontaneously leukemic AK mice, in comparison with animals of this strain fed the same diet without supplements of choline and methionine.

Animals↗
PubMed1997

Altered gene expression in spontaneous hepatocellular carcinomas from male B6C3F1 mice.

In this study, we analyzed spontaneous hepatocellular carcinomas (HCCs) from male B6C3F1 mice for alterations in the expression of the genes for c-myc, insulin-like growth factor II (IGF-II), cyclin D1, transforming growth factor-alpha (TGF-alpha), and the epidermal growth factor receptor (EGFR). These genes are all important in growth control in the rodent liver, and therefore, alterations in these genes or their products may result in unregulated growth. Northern blot analysis demonstrated an increase in expression of c-myc mRNA in five of 21 (24%) spontaneous HCCs compared with nontumor tissue. Tumors that had an increase in c-myc mRNA did not have an amplified c-myc gene. Of the HCCs analyzed, 18 of 29 (62%) showed reexpression of IGF-II RNA when compared with controls. Cyclin D1 mRNA was overexpressed in seven of 27 (26%) of the tumors analyzed relative to controls. Tumors with an increase in cyclin D1 mRNA also overexpressed the cyclin D1 protein. RNA encoding for the EGFR was decreased in 21 of 23 (91%) HCCs when compared with controls. None of the 29 liver tumors analyzed for alterations in expression of TGF-alpha mRNA differed from controls. Also, each individual tumor had a unique set of molecular alterations even when different tumors from the same animal were analyzed. These novel findings suggest that IGF-II, cyclin D1. c-myc, and EGFR are important mediators of carcinogenesis in spontaneous mouse liver tumor formation.

Animals↗