Biomedical subjects

T L Goldsworthy

Publications and source records attributed to T L Goldsworthy.

At least 19 recordsLinked to original sources

PubMed2001

Topical and oral administration of the natural water-soluble antioxidant from spinach reduces the multiplicity of papillomas in the Tg.AC mouse model.

The Tg.AC mouse carrying the v-Ha-ras structural gene is a useful model for the study of chemical carcinogens, especially those acting via non-genotoxic mechanisms. This study evaluated the efficacy of the non-toxic, water-soluble antioxidant from spinach, natural antioxidant (NAO), in reducing skin papilloma induction in female hemizygous Tg.AC mice treated dermally five times over 2.5 weeks with 2.5 microg 12-O-tetradecanoylphorbol-13-acetate (TPA). The TPA-only group was considered as a control; the other two groups received, additionally, NAO topically (2 mg) or orally (100 mg/kg), 5 days/week for 5 weeks. Papilloma counts made macroscopically during the clinical observations showed a significant decrease in multiplicity (P<0.01) in the NAO topically treated group. According to histological criteria, papilloma multiplicity were lower in both topical-NAO and oral-NAO groups, but significantly so only in the oral-NAO mice (P<0.01). The beneficial effect of NAO in the Tg.AC mouse is reported.

Administration, Cutaneous↗

PubMed2001

Implications of apoptosis for toxicity, carcinogenicity, and risk assessment: fumonisin B(1) as an example.

The rates of cell proliferation and cell loss in conjunction with the differentiation status of a tissue are among the many factors contributing to carcinogenesis. Nongenotoxic (non-DNA reactive) chemicals may affect this balance by increasing proliferation through direct mitogenesis or through a regenerative response following loss of cells through cytotoxic (oncotic) or apoptotic necrosis. In a recent NTP study in Fischer rats and B6C3F(1) mice, the mycotoxin fumonisin B(1) caused renal carcinomas in male rats and liver cancer in female mice. In an earlier study in male BD-IX rats, fumonisin B(1) caused hepatic toxicity and hepatocellular carcinomas. An early effect of fumonisin B(1) exposure in these target organs is apoptosis. However, there is also some evidence of oncotic necrosis following fumonisin B(1) administration, especially in the liver. Induction of apoptosis may be a consequence of ceramide synthase inhibition and disruption of sphingolipid metabolism by fumonisin B(1). Fumonisin B(1) is not genotoxic in bacterial mutagenesis screens or in the rat liver unscheduled DNA-synthesis assay. Fumonisin B(1) may be the first example of an apparently nongenotoxic (non-DNA reactive) agent producing tumors through a mode of action involving apoptotic necrosis, atrophy, and consequent regeneration.

Africa↗

PubMed2000

Promotion by sodium barbital induces early development but does not increase the multiplicity of hereditary renal tumors in Eker rats.

Induced cell proliferation is important in the mode of action of many non-genotoxic renal carcinogens. Since Tsc2 mutant (Eker) rats are genetically predisposed to the development of renal cell tumors, they provide a useful animal model in which to study the action of renal carcinogens. Sodium barbital was used as a model non-genotoxic renal carcinogen to test whether a concentration that increased renal tubular proliferation without severe nephrotoxicity would enhance tumor induction in a hereditary tumor model. First, a subchronic concentration-response study was conducted in wild-type male Long-Evans rats to determine increased cell proliferation without severe nephrotoxicity. Rats were dosed with sodium barbital in the feed at 0, 50, 250, 500, 1000, 2000 or 4000 p.p.m. for 3 or 8 weeks. Cell proliferation within the cortex and nephrotoxicity were quantitated. Enhanced proliferation with minimal nephrotoxicity occurred at 500 p.p.m. A second study was conducted in male Tsc2 mutant rats given sodium barbital in the feed at 0, 100 or 500 p.p.m. from 9 weeks of age to either 6 or 12 months of age. An additional group of rats was treated with sodium barbital for 6 months and then provided control feed until 12 months of age. Rats necropsied at 6 months of age had a concentration-dependent increase in preneoplastic and total renal lesions. Sodium barbital-treated rats necropsied at 12 months of age had numbers of lesions that were not different from controls. Total combined preneoplastic and neoplastic lesions in the 6 month, high dose group was the same as the 12 month control group. These data show that sodium barbital caused progression to the stage of spontaneous renal lesions in Tsc2 mutant rats but did not increase their overall number. These data suggest that enhanced cell proliferation without significant cytotoxicity exerted a promotional influence in this hereditary model.

Animals↗

PubMed1999

Altered bcl-2 family expression during non-genotoxic hepatocarcinogenesis in mice.

Dysregulation of apoptosis is an important component of multistage hepatocarcinogenesis. Members of the bcl-2 protein family are important in the regulation of apoptosis and their expression is altered in several cancers. The objectives of the present study were to determine whether the expression of members of the bcl-2 protein family are altered in mouse liver during acute treatment with non-genotoxic carcinogens and throughout non-genotoxic hepatocarcinogenesis. Acute treatment of B6C3F1 mice with phenobarbital resulted in increased levels of bcl-2 and decreased levels of bax protein, while acute treatment with WY-14,643 resulted in increased bcl-2 and BAG-1 protein in the liver. Following chronic treatment, altered hepatic foci and adenomas were classified as: small-cell, heterogeneous basophilic lesions (spontaneous or tetrachlorodibenzo-p-dioxin-induced); large-cell, homogeneous basophilic lesions (WY-14,643-induced); acidophilic lesions (phenobarbital- or chlordane-induced). Of the small-cell heterogeneous basophilic lesions, 86% of foci (31/36) and 85% of adenomas (35/41) exhibited increased bcl-2 protein levels compared with surrounding normal hepatocytes, whereas only 12.5% of foci (4/36) and 12% of adenomas (5/41) exhibited increased bcl-X(L) levels. Of the large-cell, homogenous, basophilic lesions, 100% of foci (3/3) and 90% of adenomas (9/10) expressed bcl-2 protein, whereas 100% of foci (3/3) and 80% of adenomas (8/10) exhibited increased bcl-X(L) protein levels compared with surrounding normal hepatocytes. Of the acidophilic lesions, the majority of foci (28/32, 88%) and adenomas (47/50, 94%) expressed increased bcl-X(L), whereas increased bcl-2 was observed in only 12.5% of acidophilic preneoplastic foci (4/32) and 14% of acidophilic adenomas (7/50). Of the carcinomas analyzed, 81% expressed increased bcl-2 (54/67), 78% expressed increased bcl-X(L) (52/67) and 69% expressed increased levels of both bcl-2 and bcl-X(L) (46/67). Collectively, only 8% of preneoplastic foci, 3% of adenomas and 1.5% of carcinomas did not express either bcl-2 or bcl-X(L). These results suggest that regulation of apoptotic proteins is altered during non-genotoxic carcinogenesis in mouse liver. Furthermore, there were both chemical- and lesion-specific aspects of expression of apoptotic proteins during hepatocarcinogenesis in mice.

Adenoma↗

PubMed1999

Dysregulation of apoptosis by c-myc in transgenic hepatocytes and effects of growth factors and nongenotoxic carcinogens.

Regulation of apoptosis is an important component of multistage hepatocarcinogenesis. The proto-oncogene c-myc has been shown to be important in apoptosis regulation and to be amplified and overexpressed in human and rodent liver neoplasia. The objectives of the study reported here were to determine whether apoptosis regulation is altered in transgenic hepatocytes that overexpress c-myc and whether growth factors or nongenotoxic carcinogens alter apoptosis regulation in c-myc versus wild-type hepatocytes. Hepatocytes isolated from c-myc transgenic mice had four fold more c-myc RNA and protein (at 12-48 h) in addition to increased apoptosis levels compared with wild-type hepatocytes. The increased apoptosis in c-myc hepatocytes was accompanied by increased p53, bax, and bak and decreased bcl-2 protein levels. Hepatocytes overexpressing c-myc were more sensitive to apoptosis induced by bleomycin but less sensitive to apoptosis induced by transforming growth factor (TGF)-beta. Phenobarbital, a potent liver tumor promoter, inhibited apoptosis in c-myc hepatocytes but not in wild-type hepatocytes, decreased p53 and bax, and increased bcl-2 protein levels. Nafenopin inhibited apoptosis in both c-myc and wild-type hepatocytes, whereas 2,3,7,8-tetrachlorodibenzo-pdioxin did not inhibit apoptosis in either wild-type or c-myc hepatocytes. TGF-alpha inhibited apoptosis and increased bcl-X(L) and decreased bak protein levels in c-myc hepatocytes but not in wild-type hepatocytes. Insulin-like growth factor-II did not affect apoptosis in c-myc or wild-type hepatocytes. In this study, overexpression of c-myc altered the response to apoptotic stimuli in transgenic hepatocytes. Furthermore, phenobarbital and TGF-alpha inhibited c-myc-induced apoptosis, which may have resulted in a selective growth advantage for an initiated cell population and which may be a mechanism for tumor promotion.

Animals↗

PubMed1998

Methyl tertiary butyl ether-induced endocrine alterations in mice are not mediated through the estrogen receptor.

Chronic exposure to methyl tertiary butyl ether (MTBE) altered the rodent tumor incidence of endocrine-sensitive tissues and decreased the incidence of estrogen-dependent uterine cystic hyperplasia in mice. To test the hypothesis that changes in the incidence of tumors in female B6C3F1 mice after MTBE exposure are secondary to endocrine alterations, we exposed female mice to the carcinogenic dose of MTBE vapor (8000 ppm) for 3 or 21 days or 4 or 8 months under conditions similar to a previous 2-year bioassay. MTBE exposure significantly decreased body weight gain and ovary and pituitary weight at 4 and 8 months and uterine weight at all time points. After 8 months of exposure, MTBE significantly increased the length of the estrous cycle by increasing the mean number of days in both the estrus and the nonestrus stages. Histological evaluation of H&E-stained tissues showed a decrease in the number of uterine glands after subchronic MTBE exposure. DNA synthesis, as measured by the incorporation of 5-bromo-2'-deoxyuridine (BrdU), was decreased in uterine glandular and luminal epithelial cells after MTBE exposure for 3 or 21 days or 4 or 8 months. MTBE exposure decreased the number of epithelial layers in the cervix and vagina at all time points. DNA synthesis was decreased in cervical and vaginal epithelium after 21 days of MTBE. Decreased zona reticularis of adrenal glands was found after 4 and 8 months of MTBE exposure without changes in BrdU incorporation. MTBE did not competitively bind to estrogen receptor. MTBE exposure did not alter serum estrogen levels or alter the location or intensity of estrogen receptor immunoreactivity in the uterus, cervix, and vagina. These data indicate that while MTBE exposure causes multiple endocrine-related tissue and cellular responses, these effects are not mediated through the estrogen receptor.

Administration, Inhalation↗

PubMed1998

Estrogen treatment enhances hereditary renal tumor development in Eker rats.

Hormonal influences are known to affect the development of renal cell carcinoma in man and laboratory animal models. We tested the hypothesis that estrogen treatment or ovariectomy of rats modulates renal tumor development using tuberous sclerosis 2 (Tsc2) heterozygous mutant (Eker) rats in which a germline mutation predisposes the animals to renal cell tumor development. Two-month-old female wild-type and Eker rats were ovariectomized or sham-operated and treated with placebo or 5 mg 17beta-estradiol in s.c. pellets for 6 or 10 months. Rats were examined at 8 or 12 months of age, at which time the numbers of renal tumors and preneoplastic foci were quantitated and the severity of nephropathy was assessed. In contrast to what may have been expected, prolonged estrogen treatment enhanced the development of hereditary renal cell tumors, with a 2-fold greater number of preneoplastic and neoplastic renal lesions compared with untreated Eker rats. Ovariectomized Eker rats had 33% fewer renal lesions than the unmanipulated control group. No tumors or preneoplastic lesions were present in wild-type rats at either time point. Estrogen treatment increased the severity of nephropathy in both wild-type and Eker rats, whereas ovariectomy was protective against nephropathic changes. Although estrogen is not a rat renal carcinogen, it enhanced the development of hereditary renal cell tumors when administered to Eker rats. Eker rats heterozygous for a mutation in the Tsc2 locus provide a good model in which to study how genetic and hormonal factors contribute to the development of renal cell tumors and to understand the influence genetic susceptibility has on the development of renal cell carcinoma.

Animals↗

PubMed1998

Chemical transformation of mouse liver cells results in altered cyclin D-CDK protein complexes.

Dysregulated cell proliferation is one phenotypic change associated with neoplasia. Key protein complexes involved in regulating cell division are composed of cyclins, cyclin-dependent kinases (CDK) and CDK inhibitors (CDI). Many virally transformed cells in culture exhibit disrupted cyclin-CDK-CDI complexes, suggesting that such changes may play a mechanistic role in viral transformation. To determine whether similar alterations may be involved in chemical carcinogenesis we characterized cyclin D1-CDK-CDI protein complexes in a non-tumorigenic mouse liver cell line and investigated whether complexes were altered after transformation with the genotoxic carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 3-methylcholanthrene (MC). In non-tumorigenic mouse liver cells cyclin D1 associated with CDK6, CDK4 or CDK2 to form binary (cyclin D1-CDK), tertiary (cyclin D1-CDK-p27KIP1) or quaternary (cyclin D1-CDK-p21WAF1-PCNA) complexes. After chemical transformation of mouse liver cells with either MC or MNNG, select cyclin D1-CDK-CDI protein complexes were altered. In MC-transformed cells formation of various binary, tertiary and quaternary cyclin D1-CDK-(CDI) protein complexes was reduced, resulting in decreased CDK4 kinase activity. Interestingly, CDK6 kinase activity was dramatically elevated due to high levels of cyclin D3 in association with CDK6. In MNNG-transformed cells select cyclin D1-CDK6-CDI and cyclin D1-CDK2-CDI protein complexes were altered but CDK6 and CDK4 kinase activity remained unaffected. Distinct changes in cyclin D1-CDK-CDI complexes found between the two chemically transformed mouse liver cell lines suggest that each cell line harbored unique mutations or alterations that differentially contributed to stabilization of cyclin D1-CDK-CDI holoenzymes. p53 gene mutations were not detected in the MC- or MNNG-transformed mouse liver cell lines and thus were not involved in disrupting cyclin D1-CDK-CDI protein complexes. In summary, this study presents evidence that D-type CDK protein complexes can be altered physically and functionally after chemical transformation with genotoxic carcinogens, suggesting that components of the cell cycle machinery can be targeted during chemical carcinogenesis.

Animals↗

PubMed1998

Attenuation of G1 checkpoint function by the non-genotoxic carcinogen phenobarbital.

Non-genotoxic chemical carcinogens are capable of inducing tumors in rodents without interacting with or directly altering the genetic material. Since a preponderance of evidence suggests that cancer results from the accumulation of genetic alterations, the mechanisms by which many non-genotoxic carcinogens induce genotoxic events remain unclear. The present study investigated whether the mitogenic, non-genotoxic carcinogen phenobarbital (PB) could alter cell-cycle checkpoint controls, thereby indirectly leading to the accumulation of genetic damage. Initial studies involved characterizing cell-cycle checkpoint responses to DNA damage in freshly isolated B6C3F1 mouse hepatocytes. These cells responded to bleomycin-induced DNA damage by arresting in G1 and G2. Cell-cycle arrest was coupled with p53 protein induction; however, p21WAF1 protein levels remained unchanged. Studies that utilized hepatocytes isolated from C57BL p53-/- mice showed that the DNA damage-induced G1 cell-cycle arrest was dependent on p53 function, but cell-cycle arrest in G2 was not affected by loss of p53. PB was able to delay and attenuate the G1 checkpoint response without altering G2 checkpoint function. A reduction in p53 protein, but not transcript levels, was observed in hepatocytes exposed to PB. Additionally, PB delayed and attenuated p53 protein induction during DNA damage, which suggests that changes in the p53 protein may be contributing to the attenuated G1 checkpoint response caused by PB. Altered G1 checkpoint function represents an epigenetic mechanism by which phenobarbital may prevent the detection and repair of DNA damage and indirectly increase the frequency of genotoxic events above that occurring spontaneously. Abrogation of checkpoint controls may, thus, play an important mechanistic role in mitogenic, non-genotoxic chemical carcinogenesis.

Animals↗

PubMed1998

Regulation of apoptosis in mouse hepatocytes and alteration of apoptosis by nongenotoxic carcinogens.

Regulation of apoptosis is an important component of multistage hepatocarcinogenesis. The objectives of the present study were to characterize apoptosis regulation in primary mouse hepatocytes and to determine whether nongenotoxic carcinogens alter apoptosis regulation. Bleomycin-induced apoptosis was accompanied by decreases in bcl-2 and bcl-xl and increases in p53, bak, and bax protein levels. Transforming growth factor (TGF)-beta-induced apoptosis was accompanied by decreased bcl-xL and increased bak. Bleomycin-induced apoptosis was partially dependent on p53, whereas TGF-beta-induced apoptosis was independent of p53. Phenobarbital inhibited both TGF-beta and bleomycin-induced apoptosis and the normal regulation of p53, bcl-2, and bax. Nafenopin inhibited apoptosis through a mechanism dependent on PPAR-alpha and inhibited the normal regulation of bcl-2 and bak. 2,3,7,8-Tetrachlorodibenzo-p-dioxin did not alter apoptosis or its regulation. Apoptosis was increased in hepatocytes from bcl-2-null mice, which indicated that the bcl-2 family contributes to hepatocyte apoptosis regulation. This study demonstrated that apoptosis regulation in mouse hepatocytes involves distinct pathways and that diverse nongenotoxic carcinogens differentially alter molecular pathways that represent targets for hepatocarcinogenesis.

Animals↗

PubMed1997

Furan-induced liver cell proliferation and apoptosis in female B6C3F1 mice.

Furan is a potent rodent hepatocarcinogen that probably acts through non-genotoxic mechanisms involving hepatotoxicity and regenerative hepatocyte proliferation. In addition to inducing necrosis, cytotoxicants like furan may also induce cytolethality through apoptosis which has been suggested to play a key role in carcinogenesis. Hepatocyte proliferation and apoptosis were studied in female B6C3F1 mice exposed to furan by oral gavage for 3 weeks at National Toxicology Program (NTP) bioassay doses (8 and 15 mg/kg body weight) and lower (4 mg/kg). Furan treatment led to a 2- to 3-fold significant increase in liver-related enzymes and bile acids in blood serum as compared to the control group. These changes were accompanied by minor subcapsular inflammation and minimal necrosis at 8 and 15 mg furan/kg. A dose-related increase in bromodeoxyuridine-labeling index (1.4- to 1.7-fold) and hematoxylin- and eosin-defined apoptotic index (6- to 15-fold) was observed at 8 and 15 mg/kg. Co-treatment of mice with aminobenzotriazole, an irreversible inhibitor of cytochromes P-450, prevented the observed hepatotoxic effects induced by furan. These results indicate that furan elicits hepatotoxicity in a dose-related manner through a toxic metabolite and, furthermore, suggest that apoptosis is an important form of cell death at hepatocarinogenic doses under short-term conditions.

Administration, Oral↗

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↗

PubMed1997

Reverse transcription-polymerase chain reaction-based methodology to quantify differential gene expression directly from microdissected regions of frozen tissue sections.

Quantitative differences in the expression of oncogenes are a critical feature of the cancer process. Several methods are currently available for assessing differential gene expression, but none can be used to determine quantitative changes in gene expression from small numbers of cells. The ability to conduct this type of quantitative analysis would be useful in the study of definable, early stages of carcinogenesis when very few cells are involved. We therefore developed a highly sensitive, slide-based technique that incorporates the benefits of in situ polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) to quantify differential c-myc gene expression from liver tissue sections having either low or high levels of proliferating hepatocytes. To eliminate the need for isolating and quantifying mRNA, cells of interest were microdissected from frozen histological sections and their RNA directly subjected to RT-PCR amplification. These reactions were conducted in the presence of an internal RNA standard that was specifically designed to normalize differential RT and PCR efficiencies between samples. GENESCAN software analysis was used to determine the ratios of the RT-PCR products of the target gene to the RNA standard. These ratios were then normalized to the numbers of cells isolated, as quantified by image analysis, and comparative gene expression values were determined between sample groups. We conclude that this technology can be adapted to study any gene of interest in any type of frozen tissue or isolated cells. This methodology is particularly applicable to the molecular analysis of histopathologically distinct preneoplastic and neoplastic lesions identified on tissue sections.

Animals↗

PubMed1997

Perturbation of the mitosis/apoptosis balance: a fundamental mechanism in toxicology.

Perturbations of the balance between cell gain via mitosis and cell loss by apoptosis play a pivotal role in mediating and modifying the action of carcinogens and other toxicants in tissues such as liver, brain, the immune system, the gastrointestinal tract, and the reproductive organs. Apoptosis describes a highly conserved morphology associated with the death of many different cell types from diverse tissues. This symposium focused on induced changes in this critical balance as a key mechanism of action of a variety of diverse toxicants. In the colon, the "toxicology" of 5 fluorouracil (5FU) is entirely dependent on p53, since p53 knockouts lose the pathology of 5FU damage. Presumably, this is because DNA damage is not detected and there is no cell cycle arrest. In the testes, testicular germ cell survival is mediated by adjacent Sertoli cells via the Fas ligand (FasL)-Fas receptor (Fas) system. This system appears to mediate germ cell apoptosis after exposure to testicular toxicants such as the phthalate, mono(2-ethylhexyl) phthalate (MEHP). Interestingly, MEHP is a member of the peroxisome proliferator (PP) class of nongenotoxic carcinogens. PPs perturb both hepatocyte apoptosis and mitosis. This suppression of apoptosis occurs via activation of the peroxisome proliferator-activated receptor alpha (PPARalpha), providing a paradigm for the regulation of liver growth via activation of nuclear receptors. Similarly, the toxicological effects of dioxins are mediated via the Ah receptor (AHR), another ligand-activated nuclear receptor. This receptor upregulates a variety of genes (the Ah gene battery) associated with the toxicology of dioxins. Taken together, the data presented in this symposium illustrate to the toxicologist the need to quantitate and interpret modulations in apoptosis alongside more conventional assessments of S-phase. Although the toxicant may initiate cell damage, genes like Bcl-2, p53, Fas, PPARalpha, and AHR are final arbiters of the choice between death, survival, and proliferation.

Animals↗

PubMed1997

Genomic instability, as measured by microsatellite alterations, is not associated with liver tumor development in the genetically susceptible B6C3F1 mouse.

Certain human heritable forms of colon cancer have characteristically high frequencies of microsatellite alterations. These microsatellite changes are markers of genomic instability and the direct consequence of mutations in genes involved with DNA mismatch repair processes, which are in part responsible for maintaining the sequence integrity of the genome. Given that the B6C3F1 mouse is genetically predisposed to develop liver tumors we were interested in determining whether tumors derived in this strain of mouse may contain alterations in microsatellite sequences. The analysis of 48 tumors at 24 different microsatellite loci revealed that microsatellite alterations were detected in 12 of 48 tumors (25%). Although this frequency is relatively high, 11 of the 12 tumors exhibited only a single alteration and in 10 of those tumors this change was at the same microsatellite locus. Microsatellite alterations were also detected in the DNA isolated from 6 of 22 (27%) normal liver tissues with 4 of the 6 occurring at the same locus where the majority of changes were observed in the tumors. Based on these results, we conclude that the microsatellite alterations present in the mouse liver tumor tissue are most likely the result of spontaneous mutational events. Consequently, the genomic instability operational in a particular type of hereditary human colon cancer does not appear to be operational in the genetically predisposed B6C3F1 mouse liver. In addition, we demonstrated that the activation of the H-ras gene, which causes some forms of genetic instability in vitro, does not contribute to genetic instability within liver tumors as measured by microsatellite alterations.

Animals↗

PubMed1997

Loss of tumor-promoting activity of unleaded gasoline in N-nitrosodiethylamine-initiated ovariectomized B6C3F1 mouse liver.

Unleaded gasoline (UG) vapor (2056 ppm) increased the incidence of liver tumors in a chronic bioassay and exhibited tumor-promoting activity in N-nitrosodiethylamine (DEN)-initiated female mouse liver. Estrogen inhibited mouse liver tumor development and the hepatocarcinogenic and tumor-promoting dose of UG produced uterine changes suggestive of estrogen antagonism. To directly test the hypothesis that UG-induced tumor-promoting ability is secondary to its interaction with the mouse liver tumor inhibitor, estrogen, we compared the tumor-promoting ability of UG in ovariectomized (Ovex) mice with the hepatic tumor-promoting ability of UG in intact mice. Ovaries were surgically removed at 4 weeks of age. Exposure to wholly vaporized UG (2018 ppm) under bioassay and tumor-promoting conditions began at 8 weeks of age. After 4 months of exposure, UG increased relative liver weight and hepatic microsomal cytochrome P450 pentoxyresourfin-O-dealkylase and ethoxyresorufin-O-deethylase activity to a similar extent in intact and Ovex mice. Non-focal hepatocyte proliferation, as measured by the incorporation of bromo-deoxyuridine, was not changed by UG exposure and was similar in all treatment groups. After 4 months of exposure to DEN-initiated mice, UG significantly increased the volume fraction of liver occupied by foci (three-fold) as compared to control intact mice. As expected, volume of foci was elevated in DEN/Ovex/control mice as compared to DEN/intact/control mice. In DEN/Ovex mice UG did not significantly increase the focal volume fraction. Thus, the tumor promoting activity of UG, as demonstrated by increased volume fraction of liver occupied by hepatic foci in intact mice, is greatly attenuated in Ovex mice. The volume fraction data in Ovex mice support the hypothesis that the tumor promoting activity of UG is dependent upon the interaction of UG with ovarian hormones. These data also indicate that hepatic microsomal cytochrome P450 PROD and EROD induction, hepatomegaly and non-focal hepatic LI are not specific markers of hepatic tumor promoting activity of UG.

Animals↗

PubMed1997

Time course comparison of cell-cycle protein expression following partial hepatectomy and WY14,643-induced hepatic cell proliferation in F344 rats.

During recent years, there has been an extensive research focus in the area of cell-cycle control in eukaryotes and the relationship that exists between cell proliferation and cancer. The eukaryotic cell-cycle is governed by signal transduction pathways mediated by complexes of cyclin dependent kinases (CDK) and their partner cyclin proteins. This study was performed to identify differences in cell-cycle control protein expression following physical and chemical stimuli of hepatic cell growth. Protein levels of cell cycle mediators, cyclin dependent kinases (CDK 1,2,4,5), cyclin proteins (A,B,D1-D3 and E), proliferating cell nuclear antigen (PCNA), tumor suppressor proteins (p53 and Rb), and CDK inhibitory proteins (p16Ink4, p21Waf1 and p27Kip1) were examined in F344 rats following 70% partial hepatectomy or a single dose of WY14,643 over 96- and 48-h time courses, respectively. CDK1 (p34cdc2) and PCNA protein concentrations, quantified by ELISA, were significantly increased beginning at the 24-h time point and maximal at 48 h (6.9- and 3.7-fold for partial hepatectomy and 4.2- and 3.3-fold for WY14,643, respectively). Differential effects were observed with the G1 cell-cycle mediators CDK4, CDK5, and cyclin D3, p21Waf1 and p27Kip1 CDK inhibitory protein concentrations rose in accordance with the induction of DNA synthesis and histone H1 kinase activity. In addition, there were dramatic differences in p53 protein expression patterns following partial hepatectomy versus WY14,643 dosing. Because non-genotoxic hepatocarcinogens are known to induce cellular proliferation, data generated from this study may aid in elucidating the specific hepatocarcinogenic signal transduction pathways stimulated by non-genotoxic carcinogens.

Animals↗

PubMed1996

Comparative short-term effects of methyl tertiary butyl ether and unleaded gasoline vapor in female B6C3F1 mice.

PS-6 unleaded gasoline (UG) and methyl tert-butyl ether (MTBE), an UG additive, with long-term exposure at high concentrations increased liver tumors selectively in female mice. PS-6 UG is a liver tumor promoter in N-nitrosodiethylamine-initiated female mice and produces short-term effects potentially relevant to its tumor promoting ability. The new formulation of UG (91-01) and MTBE were evaluated for similar short-term effects in mouse liver. Mice were exposed to 7814 ppm MTBE, 2014 ppm 91-01 UG, or 2028 ppm PS-6 UG vapor for 3 or 21 days, 6 hr/day, 5 day/week. Relative liver weights increased and uterine weights decreased in MTBE-, 91-01 UG-, and PS-6 UG-exposed mice. Because the decrease in relative uterine weight is suggestive of hormonal modulation, we evaluated the effects of MTBE, 91-01 UG, and PS-6 UG in vivo on hepatic 17-beta estradiol metabolism in vitro. Gavage treatment with either blend of UG and with MTBE increased estrogen metabolism in isolated mouse hepatocytes. Hepatic microsomal P450 activity was assessed by 7-pentoxyresorufin-O-dealkylase (PROD) and 7-ethoxyresorufin-O-deethylase (EROD) activities. Similar increases in P450 content and PROD and EROD activities were observed in all exposed mice as compared to controls. No hepatoxicity was observed in any treatment group. The hepatic labeling index, as measured by the incorporation of 5-bromo-2'-deoxyuridine, was increased in all exposed mice at 3 days but not 21 days, indicating that MTBE and 91-01 UG are also hepatic mitogens. These data demonstrate that a newer blend of UG and the UG additive MTBE elicit short-term effects similar to those of PS-6 UG. Given that these effects are potentially related to tumor promotion and the general lack of genotoxic activity, MTBE and 91-01 UG may exhibit tumor promoting activity similar to that seen with PS-6 UG. Since the liver is under multihormonal control, the increase in hepatic estrogen metabolism and uterine effects supports a potential role for endocrine modulations in both MTBE-and UG-induced hepatocarcinogenesis.

Administration, Inhalation↗