Biomedical subjects

Bhalchandra A Diwan

Publications and source records attributed to Bhalchandra A Diwan.

32 records · Page 2Linked to original sources

PubMed2004

Micro-MRI methods to detect renal cysts in mice.

BACKGROUND: Mouse models of disease, especially using transgenic and knockout technologies, are powerful tools to analyze the molecular basis of disease. We recently reported that a new dynamic micro-MRI method with dendrimer-based contrast agents can visualize renal structure and function in normal living mice and mice with acute renal failure. While MRI contrast enhancement is useful for detecting functional impairment of the kidneys, this technology has limitations in assessing morphologic changes, particularly cystic disease, because contrast-enhanced micro-MRI depicts cysts as low-intensity areas that cannot be distinguished from fibrotic foci. METHODS: In the current study, we evaluated if micro-MRI employing a new three-dimensional MR hydrography signal sequence [three-dimensional fast imaging employing steady-state acquisition (3D-FIESTA)] can visualize chronic cystic changes without any contrast agents. RESULTS: We were able to positively depict multiple renal cortical cysts of approximately 0.2 mm diameter in a mouse model of sickle cell disease and observe serial changes of renal cysts (>0.2 mm diameter) in cyclooxygenase-2 (COX-2) knockout mice during a 21/2-month period. Some cysts decreased in size over time. CONCLUSIONS: Micro-MRI with 3D-FIESTA can depict cyst formation in the diseased kidneys of living mice without injection of contrast agents.

Anemia, Sickle Cell↗

PubMed2003

Toxicogenomic analysis of aberrant gene expression in liver tumors and nontumorous livers of adult mice exposed in utero to inorganic arsenic.

Arsenic is a known human carcinogen. We have reported that brief exposure of pregnant C3H mice to arsenite in their drinking water during gestation induced hepatocellular carcinoma (HCC) in male offspring after they became adults. Tumor formation is typically associated with multiple gene expression changes, and this study examined aberrant gene expression associated with transplacental arsenic hepatocarcinogenesis. Liver tumors and nontumorous liver samples were taken at necropsy from adult male mice exposed in utero to either 42.5 or 85 ppm arsenic as sodium arsenite or unaltered water from day 8 to 18 of gestation. Total RNA was extracted and subjected to microarray analysis. Among 600 genes, arsenic-induced HCC showed a higher rate of aberrant gene expression (>2-fold and p < 0.05, 14%) than spontaneous tumors (7.8%). Overexpression of alpha-fetoprotein, c-myc, cyclin D1, proliferation-associated protein PAG, and cytokeratin-18 were more dramatic in arsenic-induced HCC than spontaneous tumors. In nontumorous liver samples of arsenic-exposed animals, 60 genes (10%) were differentially expressed, including the increased expression of alpha-fetoprotein, c-myc, insulin-like growth factor binding protein-1, superoxide dismutase, glutathione S-transferases, and CYP2A4, and the depressed expression of CYP7B1. Real-time RT-PCR analysis largely confirmed these findings. This toxicogenomic analysis revealed several aberrant gene expression changes associated with transplacental arsenic carcinogenesis. It is indeed remarkable that expression changes occurred in adulthood even though arsenic exposure ended during gestation. Some of these aberrantly expressed genes could play a role in the development of arsenic-induced tumors, at least in the liver.

Animals↗

PubMed2003

Induction of tumors of the liver, lung, ovary and adrenal in adult mice after brief maternal gestational exposure to inorganic arsenic: promotional effects of postnatal phorbol ester exposure on hepatic and pulmonary, but not dermal cancers.

Arsenic is a recognized human carcinogen and development of rodent models remains a critically important research objective. Since gestation can be a period of high sensitivity to chemical carcinogenesis, we have performed a series of transplacental carcinogenicity studies in mice with inorganic arsenic. In this study, groups of pregnant C3H mice received drinking water containing sodium arsenite (NaAsO2) at 0, 42.5 and 85 p.p.m. arsenic ad libitum from days 8 to 18 of gestation. These doses of arsenic were well tolerated. Dams delivered normally and at weaning (4 weeks) offspring were randomly put into groups (n = 25) of males or females according to maternal dose. In an attempt to promote skin cancers initiated by transplacental arsenic, duplicate groups of control or arsenic exposed offspring were topically exposed to 12-O-tetradecanoyl phorbol-13-acetate (TPA; 2 micro g/0.1 ml acetone, twice/week) from 4 to 25 weeks of age. Irrespective of TPA exposure, male offspring showed arsenic-induced dose-related increases in hepatocellular carcinoma incidence and multiplicity, as well as increases in adrenal tumor incidence and multiplicity. In female offspring, an increase in epithelial ovarian tumors occurred with arsenic exposure regardless of TPA exposure. Females also showed pre-neoplastic lesions of the reproductive tract, including hyperplasia of the uterus and oviduct, after arsenic but independent of TPA exposure. Although TPA had no effect on skin tumors, it promoted arsenic initiated liver tumors in females and lung tumors in both sexes. Thus, inorganic arsenic, as a single agent, can consistently act as a complete transplacental carcinogen in mice, inducing tumors at multiple sites, and as a tumor initiator in some tissues. Skin tumors were not initiated by arsenic in mouse fetuses possibly indicating tissue-specific mechanisms of action. This study indicates that gestation is a period of high sensitivity to arsenic carcinogenesis.

Adrenal Gland Neoplasms↗

PubMed2003

Treatment with lovastatin, cholestyramine or niacin alters K-ras membrane association in mouse lung in a strain-dependent manner: results in females.

Hypocholesterolemic drugs may themselves increase (cholestyramine, CS) or decrease (lovastatin, Lov) peripheral tissue de novo cholesterol biosynthesis. This will alter the abundance of prenyl groups and potentially increase (CS) or decrease (Lov) K-ras membrane localization, with possible pro- or anti-carcinogenic effects (K-ras is a proto-oncogene frequently mutated in lung cancer). Female A/J, Swiss, and C57BL/6 mice were fed 2 or 4% CS, 1% niacin, or injected with Lov three (Lov-3x) or five (Lov-5x) times per week. After three weeks, serum cholesterol and triglycerides were determined enzymatically. Total, membrane, and cytoplasmic K-ras proteins were determined in lung homogenates by immunoprecipitation followed by Western blotting with a K-ras specific antibody. CS feeding increased membrane K-ras as hypothesized in A/J and C57BL/6 mice, but had no effect in Swiss mice. Lov failed in all three strains to reduce membrane K-ras, and resulted in an increase in total K-ras in A/J and C57BL/6 mice, while again lacking effect in Swiss mice. Niacin had no effect on K-ras protein in any mouse strain. These results differ from our published results for male mice of the same strains, particularly for A/J mice. Increased amounts of K-ras protein in the membrane fraction of A/J females (but not males) treated with either Lov or CS imply that if K-ras were to become mutated, CS could result in increased lung tumorigenesis and Lov would be less likely to be protective in females. In the light of these data, both sexes should be included in future animal and human chemoprevention trials.

Animals↗

PubMed2003

Transplacental carcinogenicity of inorganic arsenic in the drinking water: induction of hepatic, ovarian, pulmonary, and adrenal tumors in mice.

Arsenic is a known human carcinogen, but development of rodent models of inorganic arsenic carcinogenesis has been problematic. Since gestation is often a period of high sensitivity to chemical carcinogenesis, we performed a transplacental carcinogenicity study in mice using inorganic arsenic. Groups (n = 10) of pregnant C3H mice were given drinking water containing sodium arsenite (NaAsO(2)) at 0 (control), 42.5, and 85 ppm arsenite ad libitum from day 8 to 18 of gestation. These doses were well tolerated and body weights of the dams during gestation and of the offspring subsequent to birth were not reduced. Dams were allowed to give birth, and offspring were weaned at 4 weeks and then put into separate gender-based groups (n = 25) according to maternal exposure level. The offspring received no additional arsenic treatment. The study lasted 74 weeks in males and 90 weeks in females. A complete necropsy was performed on all mice and tissues were examined by light microscopy in a blind fashion. In male offspring, there was a marked increase in hepatocellular carcinoma incidence in a dose- related fashion (control, 12%; 42.5 ppm, 38%; 85 ppm, 61%) and in liver tumor multiplicity (tumors per liver; 5.6-fold over control at 85 ppm). In males, there was also a dose-related increase in adrenal tumor incidence and multiplicity. In female offspring, dose-related increases occurred in ovarian tumor incidence (control, 8%; 42.5 ppm, 26%; 85 ppm, 38%) and lung carcinoma incidence (control, 0%; 42.5 ppm, 4%; 85 ppm, 21%). Arsenic exposure also increased the incidence of proliferative lesions of the uterus and oviduct. These results demonstrate that oral inorganic arsenic exposure, as a single agent, can induce tumor formation in rodents and establishes inorganic arsenic as a complete transplacental carcinogen in mice. The development of this rodent model of inorganic arsenic carcinogenesis has important implications in defining the mechanism of action for this common environmental carcinogen.

Adrenal Gland Neoplasms↗

PubMed2003

JS-K, a glutathione/glutathione S-transferase-activated nitric oxide donor of the diazeniumdiolate class with potent antineoplastic activity.

We have previously shown that nitric oxide (NO) inhibits growth and induces differentiation and apoptosis in acute myeloid leukemia cells, with the HL-60 human myeloid leukemia line being particularly sensitive to NO-mediated cytolysis. With the goal of identifying a prodrug that can target NO to the leukemia cells without inducing NO-mediated systemic hypotension, we have screened a series of O(2)-aryl diazeniumdiolates designed to be stable at physiological pH but to release NO upon reaction with glutathione. O(2)-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) proved to be the most active antiproliferative agent among those tested in HL-60 cells, with an IC(50) of 0.2-0.5 microM. After 5 days of exposure to 0.5 micro M JS-K, HL-60 cells had differentiated and acquired some of the phenotypic features of normal monocytes. One- to 2-day treatment with JS-K at concentrations of 0.5-1 microM resulted in apoptosis induction in a concentration- and caspase-dependent manner. JS-K also inhibited the growth of solid tumor cell lines but to a lesser extent than HL-60 cells. JS-K was administered i.v. to nonobese diabetic-severe combined immune deficient mice at doses of up to 4 micromol/kg without inducing significant hypotension. The growth of s.c. implanted HL-60 cells was reduced by approximately 50% when the mice received i.v. injections three times/week with 4 micromol/kg boluses of JS-K. Histological examination of tumor explants from JS-K-treated animals revealed extensive necrosis. Similar results were seen with s.c. human prostate cancer (PPC-1) xenografts. Our data indicate that JS-K is a promising lead compound for the possible development of a novel class of antineoplastic agents.

Animals↗

PubMed2002

Inorganic arsenite-induced malignant transformation of human prostate epithelial cells.

Although several epidemiologic studies show an association between arsenic exposure and prostate cancer, it is still unknown whether human prostate epithelial cells are directly susceptible to arsenic-induced transformation. This study was designed to determine whether the nontumorigenic human prostate epithelial cell line RWPE-1 could be malignantly transformed in vitro by arsenite. RWPE-1 cells were continuously exposed to 5 micro M arsenite and monitored for signs of transformation, assessed as changes in matrix metalloproteinase-9 levels. After 29 weeks of exposure, the arsenite-exposed RWPE-1 cells (referred to as CAsE-PE) showed a marked increase in matrix metalloproteinase-9 secretion, a common finding in prostate malignancies. Malignant transformation was confirmed when CAsE-PE cells produced aggressive undifferentiated malignant epithelial tumors in nude mice. The tumors stained positive for human prostate-specific antigen, confirming their origin. These results are the first report of arsenite-induced malignant transformation of a human epithelial cell line and provide an important in vitro model for studying the mechanisms underlying arsenic-induced carcinogenesis in humans.

Animals↗

PubMed2002

Alterations in membrane-bound and cytoplasmic K-ras protein levels in mouse lung induced by treatment with lovastatin, cholestyramine, or niacin: effects are highly mouse strain dependent.

Agents that either increase (cholestyramine, CS) or decrease (lovastatin, Lov) de novo peripheral cholesterol synthesis may increase (CS) or decrease (Lov) ras protein membrane localization by altering protein prenylation, and potentially have pro- or anti-carcinogenic effects. Male A/J, Swiss, and C57/BL6 mice were treated with 2 or 4% CS, 1% dietary niacin, or 25mg/kg of Lov three times per week (Lov-3X) or five times per week (Lov-5X). After 3 weeks, serum cholesterol and triglycerides were determined enzymatically. Membrane and cytoplasmic K-ras proteins in lung were determined by immunoprecipitation followed by western blotting with a K-ras specific antibody. Results confirmed the hypothesis only in isolated instances. A/J mice had a significant 30% increase in cytoplasmic K-ras and a 40% decrease in membrane K-ras from Lov treatment, as predicted. C57/BL6 mice had a significant 77% increase in membrane K-ras, as expected from CS feeding. At variance with the hypothesis, Swiss mice had increased levels (3-28%) of membrane K-ras with all treatments (including Lov), and C57/BL6 mice treated with Lov had a 58-78% increase in cytoplasmic K-ras without any reduction in the levels of membrane K-ras. Niacin, predicted to have no effect on ras membrane localization, decreased cytoplasmic K-ras in A/J mice, increased both membrane and cytoplasmic K-ras in Swiss mice, and had no effect in C57/BL6 mice. Results may have differed from those predicted because of strain-dependent differences in response to the cholesterol-lowering agents. A difference in response among the mouse strains suggests that individual genetic differences may alter the effect of hypocholesterolemic agents on K-ras membrane localization, and potentially the risk of ras-dependent cancer.

Animals↗

PubMed2002

Down-regulation of von Hippel-Lindau protein in N-nitroso compound-induced rat non-clear cell renal tumors.

Non-clear cell rat kidney tumors, inducible by N-nitroso compounds but lacking mutations in the von Hippel--Lindau (VHL) coding sequence, were examined for other VHL alterations. Neither mutations nor DNA methylation was detected in a putative promoter region. By immunohistochemistry, however, VHL protein level was evidently reduced in six of the eight eosinophilic renal epithelial tumors and in all the ten nephroblastomas. Immunoblotting of normal kidney detected two VHL proteins of 20 and 22kDa in a 16-day-old fetal rat but only 20kDa protein in an adult rat. This is the first demonstration of VHL alteration at the protein level.

Adenocarcinoma↗

PubMed2002

DNA adducts and liver DNA replication in rats during chronic exposure to N-nitrosodimethylamine (NDMA) and their relationships to the dose-dependence of NDMA hepatocarcinogenesis.

Exposure of rats to the hepatocarcinogen N-nitrosodimethylamine (NDMA) (0.2-2.64 ppm in the drinking water) for up to 180 days resulted in rapid accumulation of N7- and O6-methylguanine in liver and white blood cell DNA, maximum adduct levels being reached within 1-7 days, depending on the dose. The levels of both adducts remained constant up to treatment day 28, subsequently declining slowly to about 40% of maximal levels for the liver and 60% for white blood cells by day 180. In order to elucidate the role of DNA replication in NDMA hepatocarcinogenesis, changes in liver cell labeling index (LI) were also measured on treatment days 21, 120 and 180. Although the time- and dose-dependence of the observed effects were complex, a clear trend towards increased rates of hepatocyte LI, as indicated by BrdU incorporation, with increasing NDMA doses was evident, particularly above 1 ppm, a concentration above which NDMA hepatocarcinogenicity is known to increase sharply. In contrast, no increase in Kupffer cell DNA replication was found at any of the doses employed, in accordance with the low susceptibility of these cells to NDMA-induced carcinogenesis. No significant increase in the occurrence of necrotic or apoptotic cells was noted under the treatment conditions employed. These results suggest that, in addition to the accumulation of DNA damage, alterations in hepatocyte DNA replication during the chronic NDMA exposure may influence the dose-dependence of its carcinogenic efficacy.

Animals↗

PubMed2002

Decrease in K-ras p21 and increase in Raf1 and activated Erk 1 and 2 in murine lung tumors initiated by N-nitrosodimethylamine and promoted by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Recent evidence suggests that K-ras protooncogene protein p21 may have a tumor-suppressive role in the context of development of lung adenocarcinoma. Levels of K-ras p21, raf-1, mitogen-activated protein kinases Erk 1 and 2, the phosphorylated-activated forms of Erk 1 and 2 (Erk 1P and 2P), and proliferating cell nuclear antigen (PCNA) were measured by immunoblotting in mouse lung tumors (5 to 9 mm in size) caused by N-nitrosodimethylamine (NDMA) and in control lungs. In tumors compared with normal lung, cell membrane-associated K-ras p21 was significantly decreased and cytosolic K-ras p21 increased. Total, membrane, and cytosolic raf-1 and Erk 1P and 2P were increased in tumors compared with normal lung. A single dose of 5 nmol/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) given after NDMA resulted in a significant 2.4-fold increase in tumor multiplicity. A significantly greater decrease in membrane-associated K-ras p21 and increase in total and membrane associated raf-1 occurred in the NDMA/TCDD tumors compared with the NDMA-only tumors. PCNA levels increased in tumors, a finding confirmed by immunohistochemistry, and correlated with tumor size after NDMA/TCDD treatment but not after NDMA only. The increase in raf-1 in the tumors was confirmed by immunohistochemistry, which also revealed an increase in raf-1-positive alveolar macrophages specifically associating with tumors from the earliest stages. These results suggest a possible tumor-suppressive function for K-ras p21 in lung and a positive role for raf-1 and Erk 1/2 in lung tumorigenesis. TCDD may promote tumors by contributing to downregulation of K-ras and stimulation of raf-1.

Animals↗

PubMed2002

The metallothionein-null phenotype is associated with heightened sensitivity to lead toxicity and an inability to form inclusion bodies.

Susceptibility to lead toxicity in MT-null mice and cells, lacking the major forms of the metallothionein (MT) gene, was compared to wild-type (WT) mice or cells. Male MT-null and WT mice received lead in the drinking water (0 to 4000 ppm) for 10 to 20 weeks. Lead did not alter body weight in any group. Unlike WT mice, lead-treated MT-null mice showed dose-related nephromegaly. In addition, after lead exposure renal function was significantly diminished in MT-null mice in comparison to WT mice. MT-null mice accumulated less renal lead than WT mice and did not form lead inclusion bodies, which were present in the kidneys of WT mice. In gene array analysis, renal glutathione S-transferases were up-regulated after lead in MT-null mice only. In vitro studies on fibroblast cell lines derived from MT-null and WT mice showed that MT-null cells were much more sensitive to lead cytotoxicity. MT-null cells accumulated less lead and formed no inclusion bodies. The MT-null phenotype seems to preclude lead-induced inclusion body formation and increases lead toxicity at the organ and cellular level despite reducing lead accumulation. This study reveals important roles for MT in chronic lead toxicity, lead accumulation, and inclusion body formation.

Animals↗

PubMed2002

Altered expression of G1/S regulatory genes occurs early and frequently in lung carcinogenesis in transforming growth factor-beta1 heterozygous mice.

We developed the AJBL6 transforming growth factor-beta 1 (TGF-beta1) heterozygous (HT) mouse by mating A/J mice with C57BL/6 TGF-beta1 HT mice that shows increased carcinogen-induced lung lesions with decreased latency to examine progressive events in lung tumorigenesis. Mouse cDNA macroarrays were used to identify cell cycle genes that are differentially regulated in ethyl carbamate-induced lung adenocarcinomas compared with normal lung tissue in AJBL6 TGF-beta1 HT mice using probes that were generated from tissues isolated using laser capture microdissection. While expression of the genes for cyclin D1, CDK4, and E2F1 increased in lung adenocarcinomas relative to normal lung, expression of p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), p57(Kip2), and pRb genes decreased in comparison. Competitive RT-PCR showed that the levels of cyclin D1 and CDK4 mRNAs were 2- and 3-fold higher, respectively, in lung adenocarcinomas than in normal lung, while the mRNAs for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb were 3- to 4-fold lower in adenocarcinomas than in normal lung, thus validating the macroarray findings. Competitive RT-PCR of microdissected lesions also showed that the levels of cyclin D1 and CDK4 mRNAs increased significantly, while the mRNAs for p15(Ink4b) and p27(Kip1) decreased significantly as lung tumorigenesis progressed. Immunohistochemical staining for cyclin D1 and CDK4 showed staining in >80% of nuclei in adenocarcinomas compared with fewer than 20% of nuclei staining positively in normal lung. In contrast, while >60% of normal lung cells showed immunostaining for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb, staining for these proteins decreased in hyperplasias, adenomas, and adenocarcinomas. These data show that multiple components of the cyclin D1/CDK4/p16(Ink4a)/pRb signaling pathway are frequently altered early in lung lesions of AJBL6 TGF-beta1 HT mice that are induced by ethyl carbamate as a function of progressive lung carcinogenesis, suggesting that components of this pathway may be potential targets for gene therapy.

Adenocarcinoma↗

PubMedDate not supplied

Metallothionein-I/II double knockout mice are no more sensitive to the carcinogenic effects of nickel subsulfide than wild-type mice.

Metallothionein (MT) is a high-affinity metal-binding protein thought to mitigate the toxicity of various metals. MT may limit the toxicity of a metal by direct binding or through action as an antioxidant for metals that generate reactive oxygen species. Nickel compounds have carcinogenic potential in humans and animals, possibly by production of oxidative stress. The impact of MT deficiency on the carcinogenic effects of nickel is unknown. Thus, groups (n=25) of male MT-I/II double knockout (MT-null) or MT wild-type (WT) mice were exposed to a single treatment of nickel (0.5 or 1.0 mg Ni3S2/site, intramuscularly, [i.m.], into both hind legs), or left untreated (control) and observed over the next 104 weeks. There were no differences in the incidence of spontaneous tumors in MT-null and WT mice. Nickel induced injection site fibrosarcomas in a dose-related fashion to a similar extent in both WT and MT-null mice. Nickel-treatment had no effect on total lung tumor incidence, although some phenotypic-specific differences occurred in the proportion of benign and malignant pulmonary tumors. Overall, MT-null mice appear no more sensitive to the carcinogenic effects of nickel than WT mice. Thus, poor MT production does not appear to be a predisposing factor for nickel carcinogenesis.

Animals↗