[Show abstract][Hide abstract] ABSTRACT: Chronic exposure to high levels of inorganic arsenic (iAs) has been associated with cancerous and non-cancerous health effects, including cardiovascular effects. However, the mechanism for a presumed toxic effect of arsenic on vascular tissue is not clear. Our working hypothesis is that inorganic trivalent arsenic and its methylated metabolites react with cysteine-containing cellular proteins and alter their function leading to adverse events such as cytotoxicity or proliferation. In this study, human microvascular endothelial cells (HMEC1) and mouse microvascular endothelial cells (MFP-MVEC) were exposed to arsenite (iAsIII), monomethylarsonous acid (MMAIII), or dimethylarsinous acid (DMAIII) for 72 h to evaluate cytotoxicity, and for 24, 48 or 72 h to evaluate cell proliferation. Both cell lines showed similar LC50 values, from 0.1 to 2.4 μM, for all three trivalent arsenicals. The endothelial cells treated with1 nM to 1 μM concentrations of the three trivalent arsenicals did not show increased cell proliferation at 24, 48 or 72 h or increased rate of proliferation at 72 h of exposure. Overall, cytotoxicity of trivalent arsenicals to microvascular endothelial cells is similar to their cytotoxicity to epithelial cells, and that these compounds are not mitogenic.
[Show abstract][Hide abstract] ABSTRACT: Tobacco smoking is a major risk factor for multiple human cancers including urinary bladder carcinoma. Tobacco smoke is a complex mixture containing chemicals that are known carcinogens in humans and/or animals. Aromatic amines a major class of DNA-reactive carcinogens in cigarette smoke, are not present at sufficiently high levels to fully explain the incidence of bladder cancer in cigarette smokers. Other agents in tobacco smoke could be excreted in urine and enhance the carcinogenic process by increasing urothelial cell proliferation. Nicotine is one such major component, as it has been shown to induce cell proliferation in multiple cell types in vitro. However, in vivo evidence specifically for the urothelium is lacking. We previously showed that cigarette smoke induces increased urothelial cell proliferation in mice. In the present study, urothelial proliferative and cytotoxic effects were examined after nicotine treatment in mice and rats. Nicotine hydrogen tartrate was administered in drinking water to rats (52ppm nicotine) and mice (514ppm nicotine) for 4 weeks and urothelial changes were evaluated. Histopathologically, 7/10 rats and 4/10 mice showed simple hyperplasia following nicotine treatment compared to none in the controls. Rats had an increased mean BrdU labeling index compared to controls, although it was not statistically significantly elevated in either species. Scanning electron microscopic visualization of the urothelium did not reveal significant cytotoxicity. These findings suggest that oral nicotine administration induced urothelial hyperplasia (increased cell proliferation), possibly due to a mitogenic effect of nicotine and/or its metabolites.
[Show abstract][Hide abstract] ABSTRACT: Diuron is carcinogenic to the rat urinary bladder at high dietary levels. The proposed mode of action (MOA) for diuron is urothelial cytotoxicity and necrosis followed by regenerative urothelial hyperplasia. Diuron-induced urothelial cytotoxicity is not due to urinary solids. Diuron is extensively metabolized, and in rats, N-(3,4-dichlorophenyl)urea (DCPU) and 4,5-dichloro-2-hydroxyphenyl urea (2-OH-DCPU) were the predominant urinary metabolites; lesser metabolites included N-(3,4-dichlorophenyl)-3-methylurea (DCPMU) and trace levels of 3,4-dichloroaniline (DCA). In humans, DCPMU and DCPU have been found in the urine after a case of product abuse. To aid in elucidating the MOA of diuron and to evaluate the metabolites that are responsible for the diuron toxicity in the bladder epithelium, we investigated the urinary concentrations of metabolites in male Wistar rats treated with 2500ppm of diuron, the urothelial cytotoxicity in vitro of the metabolites and their gene expression profiles. DCPU was found in rat urine at concentrations substantially greater than the in vitro IC50 and induced more gene expression alterations than the other metabolites tested. 2-OH-DCPU was present in urine at a concentration approximately half of the in vitro IC50, whereas DCPMU and DCA were present in urine at concentrations well below the IC50. For the diuron-induced MOA for the rat bladder, we suggest that DCPU is the primary metabolite responsible for the urothelial cytotoxicity with some contribution also by 2-OH-DCPU. This study supports a MOA for diuron-induced bladder effects in rats consisting of metabolism to DCPU (and 2-OH-DCPU to a lesser extent), concentration and excretion in urine, urothelial cytotoxicity, and regenerative proliferation.
[Show abstract][Hide abstract] ABSTRACT: Inorganic arsenic (iAs) is a known human carcinogen at high exposures, increasing the incidences of urinary bladder, skin and lung cancers. In most mammalian species, ingested iAs is excreted mainly through urine primarily as dimethylarsinic acid (DMA(V)). In wild type (WT) mice, iAs, DMA(V) and dimethylarsinous acid (DMA(III)) (iAs(III)) exposure induces formation of intramitochondrial urothelial inclusions. Arsenite (iAs(III)) also induced intranuclear inclusions in arsenic (+3 oxidation state) methyltransferase knockout (As3mt KO) mice. The arsenic induced formation of inclusions in the mouse urothelium was dose and time dependent. The inclusions do not occur in iAs-treated rats, and do not appear to be related to arsenic-induced urothelial cytotoxicity. Similar inclusions in exfoliated urothelial cells from humans exposed to iAs have been incorrectly identified as micronuclei. We have characterized the urothelial inclusions using transmission electron microscopy (TEM), DNA-specific 4',6-diamidino-2-phenylindole (DAPI) and non-DNA-specific Giemsa staining, and determined the arsenical content. The mouse inclusions stained with Giemsa but not with the DAPI stain. Analysis of urothelial mitochondrial and nuclear enriched fractions isolated from WT (C57BL/6) and As3mt KO mice exposed to iAs(V) for 4 weeks showed higher levels of of iAs(V) in the treated groups. iAs(III) was the major arsenical present in the enriched nuclear fraction from iAs(V)-treated As3mt KO mice. In conclusion, the urothelial cell inclusions induced by arsenicals appears to serve as a detoxifying sequestration mechanism similar to other metals and they do not represent micronuclei.
[Show abstract][Hide abstract] ABSTRACT: Inorganic arsenic (arsenite and arsenate) at high exposures is a known human carcinogen, inducing tumors of the urinary bladder, skin, and lungs. In two experiments, we examined the urothelial proliferative effects of treatment with 173 ppm sodium arsenite (100 ppm arsenic) in the drinking water for 6 and 24 hr, and 3, 7, and 14 days in female F344 rats and 43.3 ppm sodium arsenite (25 ppm arsenic) in female C57BL/6 wild-type and arsenic (+3 oxidation state) methyltransferase knockout (As3mt KO) mice that are unable to methylate arsenicals. In the rat and both mouse genotypes, scanning electron microscopy showed cytotoxic urothelial changes as early as 6 hr after the start of arsenic exposure. The severity of As(III)-induced cytotoxic urothelial changes increased over time in the rat and in the As3mt KO mouse. Light microscopy showed an increase in urothelial hyperplasia in the rat. No significant increases in bromodeoxyuridine-labeling index were observed. The data support the hypothesis that the sequence of events in the mode of action for urothelial effects of orally administered inorganic arsenic in the rat and mouse involves superficial cytotoxicity with consequent regenerative increased cell proliferation similar to the findings associated with the administration of dimethylarsinic acid (DMA(V)) in rats.
[Show abstract][Hide abstract] ABSTRACT: Chronic exposure to inorganic arsenic (iAs) is carcinogenic to the human urinary bladder. It produces urothelial cytotoxicity and proliferation in rats and mice. DMA(V), a major methylated urinary metabolite of iAs, is a rat bladder carcinogen, but without effects on the mouse urothelium. DMA(III) was shown to be the likely urinary metabolite of DMA(V)inducing urothelial changes and is also postulated be one of the active metabolites of iAs. To evaluate potential DMA(III)-induced urothelial effects, it was administered to As3mt knockout mice which cannot methylate arsenicals. Female C57BL/6 wild type and As3mt knockout mice (10/group) were administered DMA(III), 77.3ppm in water for four weeks. Urothelial effects were evaluated by light and scanning electron microscopy (EM) and immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation. EM findings were rated 1 to 5, with higher rating indicating greater extent of cytotoxicity visualized. DMA(III) significantly increased the BrdU labeling index, a ratio of BrdU labeled cells to non-labeled cells, in the treated knockout group compared to control and wild type treated groups. DMA(III) induced simple hyperplasia in more knockout mice (4/10) compared to wild type mice (2/10). All treated knockout mice had more and larger intracytoplasmic granules compared to the treated wild type mice. Changes in EM classification were not significant. In conclusion, DMA(III) induces urothelial toxicity and regenerative hyperplasia in mice and most likely plays a role in inorganic arsenic-induced urothelial changes. However, DMA(V) does not induce hyperplasia in mice, suggesting that urinary concentrations of DMA(III) do not reach cytotoxic levels in DMA(V)-treated mice.
[Show abstract][Hide abstract] ABSTRACT: It is unclear whether the process of spontaneous and chemically induced hemangiosarcoma and hemangioma formation in mice involves the transformation of differentiated endothelial cells (ECs) or recruitment of multipotential bone marrow-derived hematopoietic stem cells or endothelial progenitor cells (EPCs), which show some degree of endothelial differentiation. In the present study, immunohistochemical staining for hematopoietic stem cell markers (CD45 and CD34), EC markers (vascular endothelial growth factor receptor 2 [VEGFR2], CD31, and factor VIII-related antigen), and a myeloid lineage marker (CD14) was employed to better define the origin of hemangiosarcomas and hemangiomas in mice. Staining was negative for CD45, factor VIII-related antigen, and CD14 and positive for CD34, VEGFR2, and CD31, indicating that mouse hemangiosarcomas and hemangiomas are composed of cells derived from EPCs expressing CD34, VEGFR2, and CD31 but not factor VIII-related antigen. The lack of CD45 expression suggests that mouse vascular tumors may arise from EPCs that are at a stage later than hematopoietic stem cells. Since factor VIII-related antigen expression is known to occur later than CD31 expression in EPCs, our observations may indicate that these tumor cells are arrested at a stage prior to complete differentiation. In addition, myeloid lineage cells do not appear to contribute to hemangiosarcoma and hemangioma formation in mice.
[Show abstract][Hide abstract] ABSTRACT: Diuron, a substituted urea herbicide, is carcinogenic to the rat urinary bladder at high dietary levels (2500 ppm). To further elucidate the mode of action, this study aimed to determine the time course and sequence of bladder cytotoxic and proliferative changes induced by diuron treatment of male Wistar rats. Rats were randomized into 2 groups (control and 2500 ppm diuron) and treated for 28 days. Ten rats from each group were terminated on each of study days 1, 3, 7, or 28. Scanning electron microscopy (SEM) showed urothelial cell swelling beginning on day 1, and by day 28, showed extensive necrosis, exfoliation and piling up of cells suggestive of hyperplasia. No difference in the BrdU labeling index was detected. In a second experiment, rats were randomized into control and diuron-treated groups and treated for 7 days or 8 weeks. After 7 days, transmission electron microscopy (TEM) showed cell degenerative changes and distention of the cytoplasm, organelles and nuclei characteristic of cytolysis. This resulted in protrusion of the superficial cells into the lumen, corresponding to the cell swelling observed previously by SEM. After 8 weeks, bladders in the diuron treated group showed an increased incidence of simple hyperplasia by light microscopy (6/10, p <0.05) compared to controls (0/10) and a significantly different SEM classification. In summary, our results support the hypothesis that urothelial cytotoxicity followed by regenerative cell proliferation are the sequential key events that occur with high dose diuron exposure in rats.
[Show abstract][Hide abstract] ABSTRACT: Inorganic arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is metabolized to organic methylated arsenicals. 2,3-Dimercaptopropane-1-sulfonic acid (DMPS), a chelating agent, is capable of reducing pentavalent arsenicals to the trivalent state and binding to the trivalent species, and it has been used in the treatment of heavy metal poisoning in humans. Therefore, we investigated the ability of DMPS to inhibit the cytotoxicity and regenerative urothelial cell proliferation induced by arsenate administration in vivo. Female rats were treated for 4 weeks with 100 ppm As(V). DMPS (2800 ppm) co-administered in the diet significantly reduced the As(V)-induced cytotoxicity of superficial cells detected by scanning electron microscopy (SEM), and the incidence of simple hyperplasia observed by light microscopy and the bromodeoxyuridine (BrdU) labeling index. It also reduced the total concentration of arsenicals in the urine and the methylation of arsenic. There were no differences in oxidative stress as assessed by immunohistochemical staining for 8-hydroxy-2'-deoxyguanosine (8OHdG) of the bladder urothelium. No differences were detected in urine sediments between groups. These data suggest that DMPS has the ability to inhibit both arsenate-induced acute toxicity and regenerative proliferation of the rat bladder epithelium, most likely by decreasing exposure of the urothelium to trivalent arsenicals excreted in the urine. These data provide additional evidence that the effects of arsenate exposure in vivo do not appear to be related to oxidative effects on dG in DNA.
[Show abstract][Hide abstract] ABSTRACT: Essential oils from mint plants, including peppermint and pennyroyal oils, are used at low levels as flavoring agents in various foods and beverages. Pulegone is a component of these oils. In a 2-year bioassay, oral administration of pulegone slightly increased the urothelial tumor incidence in female rats. We hypothesized that its mode of action (MOA) involved urothelial cytotoxicity and increased cell proliferation, ultimately leading to tumors. Pulegone was administered by gavage at 0, 75, or 150 mg/kg body weight to female rats for 4 and 6 weeks. Fresh void urine and 18-h urine were collected for crystal and metabolite analyses. Urinary bladders were evaluated by light microscopy and scanning electron microscopy (SEM) and bromodeoxyuridine (BrdU) labeling index. Pulegone and its metabolites, piperitenone, piperitone, menthofuran, and menthone, were tested for cytotoxicity in rat (MYP3) and human (1T1) urothelial cells by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. No abnormal urinary crystals were observed by light microscopy. Urine samples (18-h) showed the presence of pulegone, piperitone, piperitenone, and menthofuran in both treated groups. By SEM, bladders from treated rats showed superficial necrosis and exfoliation. There was a significant increase in the BrdU labeling index in the high-dose group. In vitro studies indicated that pulegone and its metabolites, especially piperitenone, are excreted and concentrated in the urine at cytotoxic levels when pulegone is administered at high doses to female rats. The present study supports the hypothesis that cytotoxicity followed by regenerative cell proliferation is the MOA for pulegone-induced urothelial tumors in female rats.
[Show abstract][Hide abstract] ABSTRACT: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists and PPARγ/α dual agonists are used in the treatment of type 2 diabetes mellitus and hyperlipidemias. In carcinogenicity studies, some of these agonists induced hemangiomas/hemangiosarcomas in mice, but not in rats. We hypothesized that increased endothelial cell (EC) proliferation may be involved in the mechanism of PPAR agonist-induced vascular tumors in mice. We previously showed that the sarcomagenic PPARγ agonist troglitazone (TG) increased EC proliferation in brown and white adipose tissue and liver in mice at sarcomagenic doses (400 and 800 mg/kg) after four weeks of treatment. In vitro, TG had a mitogenic effect on mouse microvascular mouse ECs by increasing cell proliferation and survival. The current studies showed that treatment of mouse ECs in vitro induced alterations in proliferation pathway gene expression, especially the expression of insulin-like growth factor-1, but had no effect on mouse oxidative stress pathways. In vivo, treatment with vitamin E did not inhibit TG-induced EC proliferation in liver and adipose tissue. In addition, no hypoxic effect was detected in adipose tissue of TG-treated mice; however, TG had a minor effect on hepatocellular hypoxia. These results provide additional evidence supporting a direct mitogenic effect in the mode of action of TG-induced hemangiosarcomas in mice.
[Show abstract][Hide abstract] ABSTRACT: The PPARγ agonist troglitazone (TG) induced an increased incidence of hemangiosarcomas in mice but was not carcinogenic in rats. In contrast, pioglitazone (PIO) did not induce hemangiosarcomas or any other tumors in mice. We previously demonstrated that TG increased the proliferation of endothelial cells (ECs) in liver and adipose tissue in mice, and acted as a mitogenic stimulant and an inhibitor of apoptosis in vitro in mouse, but not human, ECs. In the present study, we investigated whether TG had any effect on the proliferation of ECs in rats. We also evaluated the in vivo and in vitro effects of PIO on ECs in mice. In rats, TG did not increase the Ki-67 labeling index (LI) of ECs in liver or adipose tissue at doses used in the two-year bioassay, and did not increase hepatocyte proliferation. PIO administered to mice did not increase the Ki-67 LI of hepatocytes or ECs in liver or white adipose tissue, but slightly increased the EC proliferation in brown adipose tissue. PIO was slightly mitogenic on cultured mouse ECs after 3 days of treatment but not after 6 days, and there was no inhibition of apoptosis, in contrast to what was seen with TG. The data support the conclusion that sustained EC proliferation in mice is necessary, for the induction of hemangiosarcomas by TG, and these short-term and long-term effects are not seen with TG in the rat or with PIO in mice, treatments that also are not related to the induction of hemangiosarcomas in two-year bioassays.
[Show abstract][Hide abstract] ABSTRACT: Transfluthrin, a pyrethroid insecticide, induced urinary bladder tumors in rats but not in mice in 2-year bioassays. We investigated the urothelial effects of transfluthrin in vivo in rats and the effects of its major metabolite tetrafluorobenzoic acid (TFBA) in vitro on rat (MYP3) and human (1T1) urothelial cell lines. Rats were fed diet containing 0, 2000 or 5000 (with and without 1.25% NH(4)Cl) ppm transfluthrin for 4 weeks or 0 or 2000 ppm transfluthrin for 13 weeks. After 4 weeks, there was no evidence of hyperplasia or increased proliferation in any treatment group. After 13 weeks treatment with 2000 ppm, cytotoxicity and necrosis of the rat urothelial superficial layer were detected by scanning electron microscopy. The urinary concentration of TFBA in rats fed 2000 ppm transfluthrin was 2.94±0.67 mM. The LC(50) of TFBA was 2.25 mM for MYP3 cells and 2.43 mM for 1T1 cells. These studies support cytotoxicity and regenerative proliferation as the mechanism for induction of bladder tumors with high oral doses of transfluthrin due to metabolism of transfluthrin to the weakly cytotoxic TFBA which is excreted at high concentrations in the urine of rats administered high doses of transfluthrin (≥2000 ppm) for an extended period.
Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 03/2011; 49(6):1215-23. DOI:10.1016/j.fct.2011.02.022 · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The enzyme arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes reactions converting inorganic arsenic to methylated metabolites, some of which are highly cytotoxic. In a previous study, female As3mt knockout (KO) mice treated with diet containing 100 or 150 ppm arsenic as arsenite showed systemic toxicity and significant effects on the urothelium. In the present study, we showed that the cytotoxic and proliferative effects of arsenite administration on the urothelium are dose dependent. Female wild-type C57BL/6 mice and As3mt KO mice were divided into five groups (n = 7) with free access to drinking water containing 0, 1, 10, 25, or 50 ppm arsenic as arsenite for 4 weeks. At sacrifice, urinary bladders of both As3mt KO and wild-type mice showed hyperplasia by light microscopy; however, the hyperplasia was more severe in the As3mt KO mice. Intracytoplasmic granules were detected in the urothelium of As3mt KO and wild-type mice at arsenic doses ≥ 10 ppm but were more numerous, more extensive, and larger in the KO mice. A no effect level for urothelial effects was identified at 1 ppm arsenic in the wild-type and As3mt KO mice. In As3mt KO mice, livers showed mild acute inflammation and kidneys showed hydronephrosis. The present study shows a dose-response for the effects of orally administered arsenite on the bladder urothelium of wild-type and As3mt KO mice, with greater effects in the KO strain but with a no effect level of 1 ppm for both.
[Show abstract][Hide abstract] ABSTRACT: Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in drinking water is carcinogenic to humans, inducing skin, urinary bladder and lung tumors. In vivo, inorganic arsenic is metabolized to organic methylated arsenicals including the highly toxic dimethylarsinous acid (DMA(III)) and monomethylarsonous acid (MMA(III)). Short-term treatment of rats with 100 microg/g trivalent arsenic (As(III)) as sodium arsenite in the diet or in drinking water induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation and hyperplasia. The objectives of this study were to determine if these arsenic-induced urothelial effects are dose responsive, the dose of arsenic at which urothelial effects are not detected, and the urinary concentrations of the arsenical metabolites. We treated female F344 rats for 5 weeks with sodium arsenite at dietary doses of 0, 1, 10, 25, 50, and 100 ppm. Cytotoxicity, cell proliferation and hyperplasia of urothelial superficial cells were increased in a dose-responsive manner, with maximum effects found at 50 ppm As(III). There were no effects at 1 ppm As(III). The main urinary arsenical in As(III)-treated rats was the organic arsenical dimethylarsinic acid (DMA(V)). The thio-metabolites dimethylmonothioarsinic acid (DMMTA(V)) and monomethylmonothioarsinic acid (MMMTA(V)) were also found in the urine of As(III)-treated rats. The LC(50) concentrations of DMMTA(V) for rat and human urothelial cells in vitro were similar to trivalent oxygen-containing arsenicals. These data suggest that dietary As(III)-induced urothelial cytotoxicity and proliferation are dose responsive, and the urothelial effects have a threshold corresponding to the urinary excretion of measurable reactive metabolites.