Biomedical subjects

Brett T Spear

Publications and source records attributed to Brett T Spear.

15 recordsLinked to original sources

PubMed2006

PCB 153, a non-dioxin-like tumor promoter, selects for beta-catenin (Catnb)-mutated mouse liver tumors.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental toxicants which act as liver tumor promoters in rodents and can be classified as either dioxin-like or non-dioxin (phenobarbital [PB])-like inducers of cytochrome P-450. Since we have previously shown that tumor promotion by PB leads to clonal outgrowth of beta-catenin (Catnb)-mutated but not Ha-ras-mutated mouse liver tumors, we were interested to know whether the non-dioxin-like tumor promoter 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) shows the same selective pressure during tumor promotion. Male B6129SF2/J mice were given a single injection of N-nitrosodiethylamine (90 mg/kg body weight) at 9 weeks of age, followed by 39 weeks of treatment with PCB 153 (20 biweekly ip injections of 300 mumol/kg body weight) or corn oil as a control. Animals were killed 15 weeks after the last PCB 153 injection and liver tumors were identified by immunohistochemical staining of glutamine synthetase (GS) and analyzed for Catnb, Ha-ras, and B-raf mutations. Quantitative analyses revealed that GS-positive tumors were much larger and more frequent in livers from PCB 153-treated mice than in control animals, whereas GS-negative tumors were similar in both groups. Almost 90% (34/38) of all tumors from PCB 153-treated animals contained Catnb mutations, which compares to approximately 45% (17/37) of tumors in the control group. Ha-ras- and B-raf-mutated liver tumors were rare and not significantly different between treatment groups. These results clearly indicate that PCB 153 strongly selects for Catnb-mutated, GS-positive liver tumors, which is similar to the known action of PB, a prototypical tumor promoter in rodent liver.

Animals↗

PubMed2006

Inhibition of hepatocarcinogenesis by the deletion of the p50 subunit of NF-kappaB in mice administered the peroxisome proliferator Wy-14,643.

Wy-14,643 (WY) is a hypolipidemic drug that induces hepatic peroxisome proliferation and tumors in rodents. We previously showed that peroxisome proliferators increase NF-kappaB DNA binding activity in rats, mice, and hepatoma cell lines, and that mice deficient in the p50 subunit of NF-kappaB had much lower cell proliferation in response to the peroxisome proliferator ciprofibrate. In this study we examined the promotion of hepatocarcinogenesis by WY in the p50 knockout (-/-) mice. The p50 -/- and wild type mice were first administered diethylnitrosamine (DEN) as an initiating agent. Mice were then fed a control diet or a diet containing 0.05% WY for 38 weeks. Wild-type mice receiving DEN only developed a low incidence of tumors, and the majority of wild-type mice receiving both DEN and WY developed tumors. However, no tumors were seen in any of the p50 -/- mice. Cell proliferation and apoptosis were measured in hepatocytes by BrdU labeling and the TUNEL assay, respectively. Treatment with DEN + WY increased both cell proliferation and apoptosis in both the wild-type and p50 -/- mice; DEN treatment alone has no effect. In the DEN/WY-treated mice, cell proliferation and apoptosis were slightly lower in the p50 -/- mice than in the wild-type mice. These data demonstrate that NF-kappaB is involved in the promotion of hepatic tumors by the peroxisome proliferator WY; however, the difference in tumor incidence could not be attributed to alterations in either cell proliferation or apoptosis.

Acyl-CoA Oxidase↗

PubMed2005

Role of oxidative stress in peroxisome proliferator-mediated carcinogenesis.

In this review, the evidence about the role of oxidative stress in the induction of hepatocellular carcinomas by peroxisome proliferators is examined. The activation of PPAR-alpha by peroxisome proliferators in rats and mice may produce oxidative stress, due to the induction of enzymes like fatty acyl coenzyme A (CoA) oxidase (AOX) and cytochrome P-450 4A1. The effect of peroxisome proliferators on the antioxidant defense system is reviewed, as is the effect on endpoints resulting from oxidative stress that may be important in carcinogenesis, such as lipid peroxidation, oxidative DNA damage, and transcription factor activation. Peroxisome proliferators clearly inhibit several enzymes in the antioxidant defense system, but studies examining effects on lipid peroxidation and oxidative DNA damage are conflicting. There is a profound species difference in the induction of hepatocellular carcinomas by peroxisome proliferators, with rats and mice being sensitive, whereas species such as nonhuman primates and guinea pigs are not susceptible to the effects of peroxisome proliferators. The possible role of oxidative stress in these species differences is also reviewed. Overall, peroxisome proliferators produce changes in oxidative stress, but whether these changes are important in the carcinogenic process is not clear at this time.

Animals↗

PubMed2005

Dietary vitamin E does not inhibit the promotion of liver carcinogenesis by polychlorinated biphenyls in rats.

In this study, the effect of dietary vitamin E on the hepatic tumor-promoting activity of PCB-77 and PCB-153 in female Sprague-Dawley rats (175-200 g) was investigated. One week after diethylnitrosamine injection, rats were fed purified diets containing 10, 50, or 250 mg/kg vitamin E in the form of alpha-tocopheryl acetate. Starting 1 wk later, we injected rats i.p. with vehicle (corn oil) or PCB-77 or PCB-153 (300 mumol/kg) every 14 d for 4 injections. All rats were killed 10 d after the last PCB injection. The number and volume of placental glutathione S-transferase (PGST)-positive foci were increased by PCB-77 but not by PCB-153. Vitamin E did not affect the induction of PGST-positive foci. PCB-77, but not PCB-153, increased hepatic NF-kappaB activity. In conclusion, dietary vitamin E supplementation does not protect against the induction of altered hepatic focal lesions by PCBs.

Animals↗

PubMed2004

Hereditary persistence of alpha-fetoprotein and H19 expression in liver of BALB/cJ mice is due to a retrovirus insertion in the Zhx2 gene.

The alpha-fetoprotein (AFP) and H19 genes are transcribed at high levels in the mammalian fetal liver but are rapidly repressed postnatally. This repression in the liver is controlled, at least in part, by the Afr1 gene. Afr1 was defined >25 years ago when BALB/cJ mice were found to have 5- to 20-fold higher adult serum AFP levels compared with all other mouse strains; subsequent studies showed that this elevation was due to higher Afp expression in the liver. H19, which has become a model for genomic imprinting, was identified initially in a screen for Afr1-regulated genes. The BALB/cJ allele (Afr1(b)) is recessive to the wild-type allele (Afr1(a)), consistent with the idea that Afr1 functions as a repressor. By high-resolution mapping, we identified a gene that maps to the Afr1 interval on chromosome 15 and encodes a putative zinc fingers and homeoboxes (ZHX) protein. In BALB/cJ mice, this gene contains a murine endogenous retrovirus within its first intron and produces predominantly an aberrant transcript that no longer encodes a functional protein. Liver-specific overexpression of a Zhx2 transgene restores wild-type H19 repression on a BALB/cJ background, confirming that this gene is responsible for hereditary persistence of Afp and H19 in the livers of BALB/cJ mice. Thus we have identified a genetically defined transcription factor that is involved in developmental gene silencing in mammals. We present a model to explain the liver-specific phenotype in BALB/cJ mice, even though Afr1 is a ubiquitously expressed gene.

Animals↗

PubMed2004

Effects of vitamin E on the NF-kappaB pathway in rats treated with the peroxisome proliferator, ciprofibrate.

Peroxisome proliferators (PPs) are a diverse group of nongenotoxic compounds, which induce hepatic tumors in rodents. The mechanisms leading to hepatic tumors have not been elucidated, but oxidative stress may play a role in the process. Previous studies in our laboratory have shown that peroxisome proliferators activate the transcription factor nuclear factor-kappa B (NF-kappaB) and that this activation is mediated at least in part by oxidative stress. We therefore hypothesized that increased dietary vitamin E would decrease NF-kappaB DNA binding in rodents treated with ciprofibrate (CIP). In this study, 36 male Sprague-Dawley rats were fed a purified diet containing varying levels of vitamin E (10, 50, 250 ppm alpha-tocopherol acetate). After 28 days on the purified diet, seven animals per vitamin E group received 0.01% CIP in the diet for 10 days. Electrophoretic mobility shift assays (EMSAs) showed that CIP treatment increased DNA binding of NF-kappaB. Increased dietary alpha-tocopherol acetate inhibited CIP-induced NF-kappaB DNA binding. Because NF-kappaB translocates to the nucleus upon the phosphorylation and degradation of inhibitor of IkappaB, we also used Western blots to measure cytosolic protein levels of IkappaBalpha and IkappaBbeta, and the IkappaB kinases, IKKalpha and IKKbeta. IkappaBalpha protein levels were decreased in all three CIP-treated groups, with the 10 ppm vitamin E diet also decreasing IkappaBalpha levels in control rats. No difference in IkappaBbeta protein levels was observed among any of the groups. The CIP-treated rats generally had lower protein levels of IKKalpha and IKKbeta. This study supports our working hypothesis that an increased antioxidant environment can inhibit CIP-mediated NF-kappaB induction.

Administration, Oral↗

PubMed2004

Effect of 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) on hepatocyte proliferation and apoptosis in mice deficient in the p50 subunit of the transcription factor NF-kappaB.

Polychlorinated biphenyls (PCBs) are a group of synthetic chemicals that induce and promote liver tumors in rodents. We previously showed hepatic nuclear factor kappaB (NF-kappaB) activation and increased hepatocyte proliferation in PCB-treated rats. In this study, the role of NF-kappaB in hepatocyte proliferation and apoptosis after PCB administration was analyzed in wild-type mice and in mice deficient in the NF-kappaB p50 subunit (p50-/-). In a 2-day study, mice received a single intraperitoneal (ip) injection of corn oil or PCB-153. Hepatic NF-kappaB DNA binding activity and cell proliferation were increased by PCB-153 in wild-type mice but not in p50-/- mice. In a 21-day study, mice received six ip injections of corn oil or PCB-153 (twice weekly for 3 weeks) and were euthanized 4 days after the last injection. In this study, NF-kappaB DNA binding activity was not increased after PCB-153 treatment in wild-type or p50-/- mice. Cell proliferation was significantly increased in the wild-type mice treated with PCB-153; in the p50-/- mice treated with PCB-153, cell proliferation was greater than in untreated mice but less than in wild-type mice treated with PCB-153. The livers of p50-/- mice showed greater apoptosis than those of wild-type mice; PCB-153 decreased apoptosis in p50-/- mice, with higher inhibition in the 21-day study than in the 2-day study. RNase protection assays indicated that PCB-153 decreased the mRNA level of cyclin A2, B1, B2, and C in the 2-day study, but not in the 21-day study; however, it did not affect cyclin D1 and D2 mRNA levels at either time point. Cyclin D1 protein levels were not affected by PCB-153. Taken together, these data indicate that the absence of the NF-kappaB p50 subunit alters the proliferative and apoptotic changes in mouse liver in the response to PCB-153.

Animals↗

PubMed2004

Striking differences between the mouse and the human alpha-fetoprotein enhancers.

The alpha-fetoprotein (AFP) gene is expressed abundantly in the fetal liver and transcriptionally repressed in the adult liver, but can be reactivated during liver regeneration and in liver tumors. Previous studies identified three enhancers, E1, E2, and E3, upstream of the mouse and rat Afp genes and a single enhancer upstream of the human gene. We have compared the sequences upstream of the rodent and primate AFP genes. Our analysis demonstrates that the previously identified human enhancer is the counterpart to mouse E2. This comparison also reveals that a functional primate counterpart to the rodent E1 is absent due to a deletion that removes the core region of this enhancer. Furthermore, our studies identify a novel human enhancer corresponding to rodent E3. Despite the overall similarity of E3 between human and mouse, we found differences in transcription factor binding sites between these species. A C/EBP binding site is conserved but two other motifs in rodent E3, one that binds orphan nuclear receptors and a second that binds FoxA proteins, are not conserved in humans. The human counterpart to the rodent FoxA site can bind COUP-TF factors. Despite the overall sequence similarity in E3 between mice and humans, the difference in factor binding sites in E3, as well as the absence of E1 in primates, indicates that different mechanisms regulate AFP transcription in these different species.

Animals↗

PubMed2004

FoxA proteins regulate H19 endoderm enhancer E1 and exhibit developmental changes in enhancer binding in vivo.

Multiple enhancers govern developmental and tissue-specific expression of the H19-Igf2 locus, but factors that bind these elements have not been identified. Using chromatin immunoprecipitation, we have found two FoxA binding sites in the H19 E1 enhancer. Mutating these sites diminishes E1 activity in hepatoma cells. Additional chromatin immunoprecipitations show that FoxA binds to E1 in fetal liver, where H19 is abundantly expressed, but that binding decreases in adult liver, where H19 is no longer transcribed, even though FoxA proteins are present at both times. FoxA proteins are induced when F9 embryonal carcinoma cells differentiate into visceral endoderm (VE) and parietal endoderm (PE). We show that FoxA binds E1 in VE cells, where H19 is expressed, but not in PE cells, where H19 is silent. This correlation between FoxA binding and H19 expression indicates a role for FoxA in regulating H19, including developmental activation in the yolk sac and liver and postnatal repression in the liver. This is the first demonstration of a tissue-specific factor involved in developmental control of H19 expression. These data also indicate that the presence of FoxA proteins is not sufficient for binding but that additional mechanisms must govern the accessibility of FoxA proteins to their cognate binding sites within the H19 E1 enhancer.

Acetylation↗

PubMed2003

Cell proliferation and apoptosis are altered in mice deficient in the NF-kappaB p50 subunit after treatment with the peroxisome proliferator ciprofibrate.

We previously showed that the peroxisome proliferator ciprofibrate increases hepatic NF-kappaB DNA binding activity in rats, mice, and hepatoma cell lines. Here, we analyzed the response to ciprofibrate in mice that lack the NF-kappaB p50 gene (p50-/-). Wild-type and p50-/- mice were fed a diet with or without 0.01% ciprofibrate for 10 days. NF-kappaB DNA binding activity was present and increased after ciprofibrate treatment in wild-type mice, but was not detected in p50-/- mice. The untreated p50-/- mice had a higher level of hepatic cell proliferation, as measured by BrdU labeling, than did untreated wild-type mice. However, the increase in proliferation was greater in ciprofibrate-fed wild-type mice than in ciprofibrate-fed p50-/- mice. The apoptotic index was low in wild-type mice in the presence or absence of ciprofibrate. Apoptosis was increased in untreated p50-/- mice compared to wild-type mice; apoptosis was reduced in p50-/- mice after ciprofibrate feeding. The c-Jun and JunB mRNA levels were higher in untreated p50-/- mice than in untreated control mice; c-Jun mRNA levels increased, whereas JunB mRNA levels decreased in both groups after ciprofibrate treatment. The c-Jun and JunB protein levels were the same in untreated wild-type and p50-/- mice and increased in both groups after ciprofibrate treatment. Several apoptosis-related mRNAs were higher in untreated p50-/- mice compared to untreated control mice; expression of these genes increased in both groups after ciprofibrate treatment. These data indicate that NF-kappaB contributes to the proliferative and apoptotic changes that occur in the liver in response to ciprofibrate.

Administration, Oral↗

PubMed2003

Effect of a single dose of polychlorinated biphenyls on hepatic cell proliferation and the DNA binding activity of NF-kappaB and AP-1 in rats.

Polychlorinated biphenyls (PCBs) are environmental pollutants that, because of their persistence and biomagnification, raise concerns about the health consequences of long-term exposure. PCB mixtures induce hepatocellular carcinomas in rodents, but the mechanism of their promoting activity is not clear. Previous studies have shown that oxidative stress occurs after PCB administration, with the induction of lipid peroxidation and oxidative DNA damage, which may contribute to their promoting activity. In this study, we examined whether the oxidative stress-sensitive transcription factors NF-kappaB or AP-1 were activated by PCBs in the liver. Male Sprague-Dawley rats were injected i.p. with corn oil, 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153, 30, 150, or 300 micromol/kg), 3,3',4,4'-tetrachlorobiphenyl (PCB-77, 30, 150, or 300 micromol/kg), or both PCBs (each 30 or 150 micromol/kg). Rats were euthanized 2, 6, or 24 h, or 2, 6, and 10 d after the PCB injection. Electrophoretic mobility shift assays (EMSAs) were performed to determine NF-kappaB and AP-1 DNA binding activities. The highest NF-kappaB DNA binding activity was observed in rats receiving higher doses of PCB-153 (150 and 300 micromol/kg), with peak activation occurring 2 d after injection. AP-1 activation was not detected at any timepoint. Hepatocyte proliferation, as measured by the labeling index, was increased only in groups receiving the highest dose of PCB-153 or the combination of two PCBs (150 micromol/kg each) at day 2, and not by any other PCB treatment at any timepoint. These results show that PCB-153, but not PCB-77, can induce hepatocyte proliferation and hepatic NF-kappaB activation after a single dose.

Animals↗

PubMed2002

Regulation of cell proliferation, apoptosis, and transcription factor activities during the promotion of liver carcinogenesis by polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) are environmental pollutants that are complete carcinogens and tumor promoters in the liver. The mechanisms of their promoting activities are not clear, but one possible mechanism is the induction of oxidative stress. In the present study we evaluated the ability of two PCB congeners to activate the oxidative stress-responsive transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), as well as hepatocyte cell proliferation and apoptosis, which are influenced by activation of these transcription factors, in rat liver. Two transcription factors not activated by oxidative stress, signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5), were also examined. All the animals in this study received a single dose of diethylnitrosamine (150 mg/kg) followed by four biweekly injections of 3,3',4,4'-tetrachlorobiphenyl (PCB-77) or 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) (100 or 300 micromol/kg), or both PCBs (100 micromol/kg each). Ten days after the last PCB injection, all animals were euthanized; 3 days before euthanasia all animals were implanted with Alzet osmotic pumps containing 5-bromo-2'-deoxyuridine (BrdU). The number of placental glutathione S-transferase (PGST)-positive foci were increased in rats administered PCBs, with the highest increase seen in rats administered PCB-77. The number of foci in rats administered both PCBs was intermediate between the numbers seen with either PCB-77 or PCB-153, indicating that a synergistic effect did not occur. There was a significant increase in NF-kappaB and AP-1 binding activities in hepatic nuclear extracts from rats receiving the high dose of PCB-77 or PCB-153 and in rats receiving both PCBs. In contrast, the DNA binding activities of STAT3 and STAT5 were decreased in rats administered PCBs. Cell proliferation in both focal and nonfocal hepatocytes was increased by PCB-77 but was not affected by PCB-153. Apoptotic indexes, as quantified by the TUNEL method, were increased in both focal and nonfocal hepatocytes by PCB-77 but were decreased in focal hepatocytes by PCB-153. This study shows that both PCBs alone or in combination can increase the DNA binding activities of NF-kappaB and AP-1, whereas the DNA binding activities of STAT3 and STAT5 are decreased. The induction of altered hepatic foci appears to be related to compensatory cell proliferation in PCB-77-treated rats, whereas the inhibition of apoptosis appears to be important in PCB-153-treated rats.

Animals↗

PubMed2002

The mouse alpha-fetoprotein promoter is repressed in HepG2 hepatoma cells by hepatocyte nuclear factor-3 (FOXA).

The mouse alpha-fetoprotein gene is expressed at high levels in the fetal liver and is transcriptionally silenced at birth. The repression is governed, at least in part, by the 250 base pair (bp) AFP promoter. We show here that the AFP promoter is dramatically repressed by HNF3 in HepG2 hepatoma cells. This repression is governed by the region between -205 and -150. Furthermore, this fragment can confer HNF3-mediated repression on a heterologous promoter. The repression is abolished by a mutation that is centered at -165. EMSA analyses using in vivo and in vitro synthesized proteins indicate that HNF3 proteins do not bind DNA from the -205 to -150 region. We propose that HNF3 represses AFP promoter activity through indirect mechanisms that modulate the binding or activity of a liver-enriched factor that interacts with the -165 region of the AFP promoter.

Animals↗

PubMed2002

Vitamin E inhibits hepatic NF-kappaB activation in rats administered the hepatic tumor promoter, phenobarbital.

Phenobarbital (PB) is an efficacious hepatic tumor promoter. Although the promoting activity of PB is likely related to altered cell proliferation or apoptosis, the induction of an oxidative stress environment may also be important. PB has been shown to activate the transcription factor nuclear factor-kappaB (NF-kappaB). In this study, we hypothesized that PB-induced NF-kappaB activation can be decreased by dietary vitamin E in rats. Male Sprague-Dawley rats (n = 39) were fed a purified diet with varying levels of dietary vitamin E (10, 50 or 250 mg/kg of dl-alpha-tocopherol acetate) for 28 d, at which time 8 rats per level of dietary vitamin E were fed the same diet with 500 mg/kg PB for 10 d. In the rats fed the low vitamin E diet, PB increased NF-kappaB DNA binding, but it did not affect NF-kappaB activation in rats fed higher levels of vitamin E (50 and 250 mg/kg). Vitamin E may decrease the oxidative stress created by PB by also enhancing other antioxidants; therefore, we also measured hepatic glutathione S-transferase, glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase and NAD(P)H:quinone reductase (DT-diaphorase) activities and glutathione and ascorbic acid concentrations. Increased dietary alpha-tocopherol did not affect the antioxidants and antioxidant enzymes altered by PB treatment. Thus, the effect of alpha-tocopherol acetate on NF-kappaB activation does not appear to be mediated by alterations in the antioxidant system. These results demonstrate that the activation of NF-kappaB, a transcription factor that affects cell proliferation- and apoptosis-related gene expression, can be inhibited by dietary vitamin E.

Animals↗

PubMed2002

Peroxisome proliferators do not activate the transcription factors AP-1, early growth response-1, or heat shock factors 1 and 2 in rats or hamsters.

Peroxisome proliferators (PPs) cause hepatomegaly, peroxisome proliferation, and hepatocarcinogenesis in rats and mice, whereas hamsters are less responsive to these compounds. PPs increase peroxisomal beta-oxidation and P4504A subfamily activity, which have been hypothesized to result in oxidative stress. Work in our laboratory indicated that differential modulation of the redox-sensitive transcription factor NF-kappaB may contribute to the resulting difference in species susceptibility following PP administration. Therefore, we hypothesized that other redox-sensitive transcription factors such as AP-1, early growth response gene 1 (Egr-1), and heat-shock factors 1 and 2 (HSF1/2) may also be alternatively activated in differentially susceptible species. Accordingly, we measured the activation of these transcription factors using gel mobility shift assays, with hepatic nuclear extracts derived from rats and Syrian hamsters fed two doses of three peroxisome proliferators (dibutyl-phthalate [DBP], gemfibrozil and Wy-14,643) for 6, 34, or 90 days. Although changes were observed at various time points, no consistent, dose-responsive changes were observed in the DNA binding activities of these transcription factors following PP treatment. The lack of increased binding of AP-1, Egr-1, and HSFs suggests that these factors are not involved in increased cell proliferation following PP administration, although we cannot rule out that these factors are activated at earlier time points than those examined in this study.

Animals↗