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A Buchmann

Publications and source records attributed to A Buchmann.

At least 37 records · Page 2Linked to original sources
PubMed1995

Role of cell proliferation at early stages of hepatocarcinogenesis.

Hepatocarcinogenesis in rodents is characterized by the early appearance of foci of enzyme-altered (initiated) cells which are believed to represent precursors on the way to malignancy. Proliferation of cells within enzyme-altered liver foci is generally increased as compared to surrounding normal hepatocytes. This may be mediated by changes in the rates of cell division and/or cell death (apoptosis). Cell proliferation is controlled by complex signaling networks and may be modulated by xenobiotics. In mouse--but not rat or human--liver, mutation of the Ha-ras gene appears to represent a critical genetic alteration which may confer a selective growth advantage to the mutated cells. Exogenous tumor-promoting agents may stimulate cell division and depress apoptosis of preneoplastic hepatocytes, thereby increasing the probability of cancer. By means of histochemical methods, data on the frequencies of both cell division and cell death can be collected separately and utilized for estimation of promoter efficacy. In addition, quantitative stereology may be applied to the analysis of the size distribution of enzyme-altered foci and used for modeling of hepatocarcinogenesis.

Animals↗
PubMed1995

Inhibition of apoptosis during 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated tumour promotion in rat liver.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell division and cell death (apoptosis) in glutathione S-transferase (GST-P)-positive liver foci were analyzed in diethylnitrosamine-initiated female Wistar rats that were treated with TCDD, either acutely for 3 days or chronically for 115 days. Apoptotic bodies were quantitated in liver sections simultaneously stained for GST-P expression and H&E using a novel fluorescence microscopic detection method which greatly facilitates recognition of apoptotic bodies due to their high level of eosin fluorescence. While TCDD treatment only marginally affected cell division in GST-P-positive liver foci, as estimated by 5-bromo-2'-deoxyuridine-labelling, apoptotic indices were decreased to approximately 60% and approximately 10% of control values after acute and chronic TCDD treatment, respectively. In normal liver tissue, apoptotic indices were only slightly reduced by TCDD treatment, suggesting selective inhibition of apoptosis in the enzyme-altered cell population by the dioxin. Since inhibition of apoptosis in GST-P-positive liver foci was by far more pronounced than changes in cell division, our data suggest that the promoting activity of TCDD is preferentially mediated by a decrease of apoptosis in enzyme-altered liver foci.

Animals↗
PubMed1994

Gamma-irradiation-induced micronuclei from mouse hepatoma cells accumulate high levels of the tumor suppressor protein p53.

The p53 tumor suppressor protein plays an important role in the G1 arrest of cells treated with DNA-damaging agents. Mouse hepatoma cells with wild-type or mutated p53 genotype were gamma-irradiated and the time course of p53 expression was determined by immunocytochemical staining. In p53 wild-type cells, gamma-irradiation led to a transient accumulation of the protein in the nuclei, whereas no such accumulation occurred in p53-mutated cells. Micronuclei were induced by gamma-irradiation in both wild-type and mutated cells in a dose- and time-dependent manner, but only micronuclei from p53 wild-type cells demonstrated a strongly positive staining reaction for p53 protein. This accumulation of p53 protein in micronuclei was not associated with a block in DNA synthesis as evidenced by bromodeoxyuridine labeling experiments.

Animals↗
PubMed1994

Promotion of preneoplastic foci in rat liver with 2,3,7,8-tetrachlorodibenzo-p-dioxin, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin and a defined mixture of 49 polychlorinated dibenzo-p-dioxins.

In a two-stage initiation-promotion experiment the hypothesis was investigated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TE), calculated from data of CYP1A induction in hepatocytes in primary culture, or international TCDD equivalents (ITE) are useful for evaluating the tumor-promoting potency of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) and of a defined mixture (M2) of 49 polychlorinated dibenzo-p-dioxins (PCDDs) in comparison with TCDD. Therefore, female Wistar rats were treated with an initiating dose of N-nitrosomorpholine, and subsequently received daily doses of 2, 20 and 200 ng TCDD/kg or equivalent doses of HpCDD or M2, based on TE values. After a promotion phase of 13 weeks, hepatic PCDD levels, CYP1A activity and the relative hepatic volume of adenosinetriphosphatase-negative or glutathione S-transferase P-positive preneoplastic foci were determined. After logarithmic transformation, linear PCDD level-response relationships were obtained for induction of CYP1A activity with TCDD, HpCDD or M2. Based on TE values, inducing potencies of both HpCDD and M2 were over-estimated at higher doses, whereas induction was approximately equivalent at the lowest dose. The best fit of PCDD level-response relationships of relative hepatic volumes of preneoplastic lesions was achieved using a four-parameter logistic model. Significantly different functions were calculated for promotion with TCDD or HpCDD. It is concluded that (i) different PCDD level-response relationships exist for the induction of hepatic CYP1A activity and the promotion of preneoplastic liver foci, and (ii) that TE or ITE factors provide only a rough estimate of the tumor-promoting potency of a PCDD mixture but may overestimate the risk from exposure to higher-chlorinated 2,3,7,8-substituted congeners such as HpCDD.

Animals↗
PubMed1994

Effects of 2,3,7,8-tetrachloro- and 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin on the proliferation of preneoplastic liver cells in the rat.

Using an initiation-promotion system, enzyme-altered putative preneoplastic liver foci were induced in female Wistar rats by application of diethylnitrosamine (10 mg/kg/day) for 5 days, followed by bi-weekly treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; corresponding to 100 ng/kg/day) or 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HCDD; corresponding to 5 micrograms/kg/day) for up to 17 weeks. Groups of animals were killed at various time intervals after start of promoter treatment. For evaluation of DNA synthesis, 5-bromo-2'-deoxyuridine was administered 24 h prior to killing the animals. Quantitative analysis of the number and volume fraction of adenosine-triphosphatase-deficient liver foci revealed that the promoting activity of both dioxins was roughly comparable under the experimental conditions employed. Nuclear labelling indices (LIs) of normal hepatocytes were not altered by TCDD or HCDD treatment, while a slight increase in LIs of non-parenchymal liver cells was observed. Using an immunohistochemical double-staining technique, hepatocytes within glutathione-transferase P-positive liver foci were found to show an approximately 5-to 10-fold higher LI than normal hepatocytes throughout all periods of investigation. During the time course of the experiment, LIs of foci from all treatment groups decreased with time. However, in TCDD-treated rats, and less pronounced in HCDD-treated rats, the initially high rate of proliferation persisted for a greater length of time than in non-dioxin-treated control animals. Assignment of liver foci into four transection size classes revealed that LIs in larger size classes varied considerably, indicating heterogeneity in the growth behaviour of individual liver lesions. Overall, both dioxins had no effects on the proliferation of normal hepatocytes, while LIs of enzyme-altered liver lesions were slightly enhanced by treatment with TCDD or HCDD. Whether the selective, albeit moderate increase in the proliferation of enzyme-altered liver cells is sufficient to explain the promoting activity of dioxins, or if additional factors (e.g. decrease in death rates of foci cells) are equally important, remains to be determined in further experiments.

Animals↗
PubMed1993

Immunohistochemical localization of the cytochrome P450 isozymes LMC2 and LM4B (P4501A1) in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated zebrafish (Brachydanio rerio).

Dibenzo-p-dioxins are persistent pollutants in our environment that exert a variety of biological and toxic effects in various species, in particular developmental toxicity in fish. Using zebrafish (Brachydanio rerio) as a model system, we analyzed the expression and inducibility of a constitutive isozyme (LMC2) and a dioxin-inducible isozyme (LM4B or P4501A1) of cytochrome (cyt.) P450 in different organs by immunohistochemical techniques. In untreated zebrafish, LMC2 was constitutively expressed at high levels in the liver, kidney, skin, and oral mucosa, while moderate expression was detected in gills, pseudobranch, intestine, and ovaries. LM4B was either not expressed in these organs or was found at comparatively low levels. After treatment of zebrafish with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; total dose of 15 ng per fish), LM4B expression was strongly induced in livers and kidneys and less pronounced in gills and pseudobranches, whereas no or only marginal induction was seen in skin, intestine, oral mucosa, and ovaries. In addition, cyt. P450 catalyzed 7-ethoxyresorufin-O-deethylation and phenol UDP-glucuronosyltransferase activity were also found to be inducible in liver and kidney. Our results demonstrate that zebrafish respond to inducing stimuli of TCDD in a fashion similar to that observed with related compounds in other fish species, suggesting that zebrafish may be a useful experimental model for studying biological and toxic effects of TCDD and other environmental pollutants in fish.

Animals↗
PubMed1992

p53 Mutations in human hepatocellular carcinomas from Germany.

Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas. We have examined 13 cases of human hepatocellular carcinomas from Germany for the presence of p53 aberrations in exons 4 to 8 of the gene by single-strand conformation polymorphism and restriction fragment-length polymorphism analyses and by sequencing of polymerase chain reaction products. Single base substitutions occurred in two human hepatocellular carcinomas: a C:G----T:A transition at a CpG site in codon 257, and a T:A----A:T transversion at codon 273. One of these point-mutated tumors and two additional tumors without point mutations demonstrated a loss of one p53 allele. None of the tumors was mutated in codons 12 or 61 of the c-Ha-ras gene.

Adult↗
PubMed1992

Role of mutations at codon 61 of the c-Ha-ras gene during diethylnitrosamine-induced hepatocarcinogenesis in C3H/He mice.

Liver tumors of certain strains of mice frequently contain mutations at codon 61 of the c-Ha-ras gene. In our study, we investigated the significance of these mutations in the carcinogenic process. Male C3H/He mice received a single injection of diethylnitrosamine (DEN) on day 15 after birth, and groups of animals were killed at various time intervals between 11 and 52 wk after treatment. At the earlier time points (11-29 wk), we analyzed microdissected tissue from precancerous glucose-6-phosphatase-deficient liver lesions larger than approximately 200 microns in diameter, for the presence and pattern of c-Ha-ras codon 61 mutations. In parallel, the growth characteristics of these liver lesions were studied by pulse labeling with [3H]thymidine and by determining the size distribution of the lesions. At the later time points (42-52 wk after DEN treatment), liver tumors were dissected and also analyzed for the presence of c-Ha-ras mutations. We found mutations to be already present in some of the enzyme-altered liver lesions at weeks 11-29, suggesting that the mutations occurred early in the carcinogenic process. Whereas about 10% of the precancerous focal liver lesions showed mutations in the c-Ha-ras gene, the mutation frequency was increased to about 50% in the later-appearing hepatocellular adenomas and carcinomas, suggesting that c-Ha-ras codon 61 mutations may provide a selective advantage to the mutated cell clones.

Animals↗
PubMed1992

The tumour promoters dieldrin and phenobarbital increase the frequency of c-Ha-ras wild-type, but not of c-Ha-ras mutated focal liver lesions in male C3H/He mice.

The frequency and pattern of mutations at codon 61 of the c-Ha-ras protooncogene were analysed in glucose-6-phosphatase-deficient hepatic lesions of male C3H/He mice occurring either spontaneously or after continuous treatment with 10 p.p.m. dieldrin or 500 p.p.m. phenobarbital (PB) in their diet. At 52 weeks after start of promoter administration, enzyme-altered liver lesions had developed in 41% (15/37) of untreated control mice and in 67% (10/15) and 63% (10/16) of mice treated with dieldrin or PB respectively. The average numbers of focal lesions per mouse were 0.57 in the control, 1.5 in the dieldrin and 1.0 in the PB group. Lesions were punched out from frozen liver sections and used for mutation analysis by allele-specific oligonucleotide hybridization following in vitro amplification of DNA via polymerase chain reaction. In the control group, 12 out of 21 liver lesions (57%) showed c-Ha-ras mutations, while five out of 23 (22%) and four out of 16 (25%) lesions were mutated in the dieldrin and PB groups. Taking the different numbers of animals in the three experimental groups into account, our data indicate that the tumour promoters increased the frequency of c-Ha-ras wild-type but not of c-Ha-ras mutated focal liver lesions, suggesting that the mutations had occurred spontaneously and were not related to treatment. Since c-Ha-ras mutations were found to be frequent in large but infrequent in small hepatocellular lesions, these mutations may represent in livers of C3H/He mice an endogenous promoting principle that provides a selective growth advantage to the mutated progenitor cells.

Amino Acid Sequence↗
PubMed1992

Enzyme and immunohistochemical phenotyping of diethylnitrosamine-induced liver lesions of male C3H/He, B6C3F1 and C57BL/6J mice.

Male C3H/He, B6C3F1 and C57BL/6J mice were given a single injection of diethylnitrosamine (20 micrograms/g body wt) on day 15 after birth and animals were killed 17-29 (C3H/He and B6C3F1) and 29-46 weeks (C57BL/6J) after treatment. Carcinogen-induced liver lesions were identified by a deficiency in the marker enzyme glucose-6-phosphatase and the enzymatic phenotypes of these lesions were studied by enzyme and immunohistochemical methods using serial liver sections stained for seven additional histochemical markers. In all three mouse strains, liver lesions were characterized by an increased basophilia and a decreased expression of UDP-glucuronosyl-transferase, microsomal epoxide hydrolase and NADPH-cytochrome P450 reductase, while the cytochrome P450 isoenzymes 1A2, 2C6 and 2E1 were virtually unexpressed. Quantitative analyses revealed that throughout all periods of investigation, on average greater than 70% of the glucose-6-phosphatase-deficient lesions occupying up to 99% of the total volumetric fraction expressed concomitant alterations in at least one of these additional marker stainings. Upon determination of the phenotypic complexity levels, between 70 and 90% of lesions were found to contain alterations in at least six of the markers analysed, while lesions with alterations in less than three markers were comparatively infrequent. In the light of previous observations in the rat liver system, the relative homogeneity of enzyme phenotypes and the apparent lack of time-dependent changes in enzyme expression suggest that the majority of lesions of all three mouse strains possess an increased neoplastic character already from the very early beginning of the carcinogenic process in liver.

Animals↗
PubMed1991

Progression of squamous carcinoma cells to spindle carcinomas of mouse skin is associated with an imbalance of H-ras alleles on chromosome 7.

Analysis of benign and malignant mouse skin tumors had previously shown that amplification of a mutant H-ras allele or loss of the normal allele was generally seen only in high grade or spindle cell tumors. The normal:mutant ras gene dosage has been studied directly by polymerase chain reaction amplification of DNA derived from paraffin sections of carcinomas of defined histological types. Some tumors had virtually no signal corresponding to the normal allele and these were invariably spindle cell carcinomas. In four cases where both squamous and spindle cell components could be identified within the same tumor the spindle cell component had a higher mutant:normal gene ratio. Additional experiments on cell lines derived from squamous or spindle cell tumors have demonstrated a good correlation between the ratio of normal:mutant ras and the degree of invasiveness of the cells in in vitro assays.

Alleles↗
PubMed1991

Mutational activation of the c-Ha-ras gene in liver tumors of different rodent strains: correlation with susceptibility to hepatocarcinogenesis.

The frequency and pattern of mutations at codon 61 of the c-Ha-ras gene have been analyzed in 195 liver tumors and 132 precancerous liver lesions from various rodent strains with differing susceptibility to hepatocarcinogenesis. By using the polymerase chain reaction and allele-specific oligonucleotide hybridization, C----A transversions at the first base and A----T transversions or A----G transitions at the second base of c-Ha-ras codon 61 were detected in 20-60% of spontaneous or carcinogen-induced liver tumors of the C3H/He, CBA, CF1, and B6C3F1 mouse strains, which are highly susceptible to hepatocarcinogenesis. No such mutations, however, could be found in any of the 31 liver tumors of the insensitive C57BL/6J and BALB/c mouse strains or in any of the 35 liver tumors of the comparatively resistant Wistar rat. Further analyses of c-Ha-ras codon 12 mutations in liver tumors from the three insensitive rodent strains also failed to give any positive results. In early precancerous liver lesions, c-Ha-ras codon 61 mutations were found in 13-14% of lesions of the sensitive C3H/He and B6C3F1 mouse strains but not in any of the 34 lesions of the insensitive C57BL/6J mouse. Taken together, our results indicate a close correlation between the mutational activation of the c-Ha-ras gene in liver tumors of the different rodent strains and their susceptibility to hepatocarcinogenesis, whereby the mutations appear to provide a selective growth advantage, leading to a clonal expansion of the mutated liver cell population, only in livers of sensitive but not of insensitive strains.

Animals↗
PubMed1991

Effects of polychlorinated biphenyls in rat liver: correlation between primary subcellular effects and promoting activity.

In the present study the promoting activity of various PCB and PBB isomers and congeners in rat liver has been studied and compared with a variety of primary xenobiotic-mediated enzymatic changes in this target organ. Female Wistar rats were given diethylnitrosamine (DEN; 10 mg/kg body wt for 10 days) and were subsequently treated once weekly with polychlorinated biphenyls (150 or 15 mumol/kg body wt) for a total of 8 weeks. Additional groups of rats were administered 3,3',4,4'-tetrabromobiphenyl or 3-methylcholanthrene (8 weekly injections of 15 or 150 mumol/kg body wt, respectively) or were given phenobarbital (0.05% in the diet) until the end of the experiment. Reference groups were treated with the various test compounds without prior initiation. One week and 9 weeks after cessation of promoter treatment rats were killed and the volumetric fraction of enzyme-altered foci characterized by changes in adenosine triphosphatase and gamma-glutamyl transpeptidase activity was determined as a means to quantitatively assess the extent of preneoplastic response in this organ. Out of the series of polyhalogenated biphenyls tested, promoting effects were seen with the following compounds: 2,2',4,5'-tetrachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, 2,3,4,4',5-pentachlorobiphenyl, and 3,3',4,4'-tetrabromobiphenyl, whereas no significant effects were obtained with 4-monochlorobiphenyl. In rats not treated with DEN, the two strongly promoting agents 2,3,4,4',5-pentachlorobiphenyl and 3,3',4,4'-tetrachlorobiphenyl also significantly increased the volume fraction of enzyme-altered foci over the respective controls when analyzed at the second time point of investigation. In parallel experiments, induction of liver growth and of microsomal cytochrome P450 content in liver was found to correlate well with the promoting activity of the various xenobiotics, suggesting that these parameters may be used to predict the promoting activity of polyhalogenated biphenyls in a short term assay.

Adenosine Triphosphatases↗
PubMed1991

Effects of polychlorinated biphenyls in rat liver: quantitative analysis of enzyme-altered foci.

The promotional effect of various polychlorinated biphenyls and phenobarbital on enzyme-altered lesions in the rat liver was quantified within the framework of the two-stage carcinogenesis model of Moolgavkar and colleagues. The experiment analyzed here followed an initiation-promotion protocol in which female Wistar rats were initiated with diethylnitrosamine (DEN) at 10 mg/kg body wt for 10 days followed by a 8-week period of promoter treatment with various cytochrome P450 isoenzyme inducing and noninducing compounds. This analysis included 4-monochlorobiphenyl, 2,2',4,5'-tetrachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl and 3-methylcholanthrene, all administered at 150 mumol/kg body wt, and phenobarbital which was administered continuously in the diet at 0.05% until termination. Animals were killed either 1 or 9 weeks after the end of treatment and their livers were examined for enzyme histological alterations. Focal transections were classified as falling into three phenotypic categories: ATPase dominant, GGT dominant, or ATPase plus GGT (coextensive). A quantitative method was used to analyze the data consisting of the number and sizes of the focal transections. The number of cells altered by the DEN treatment and cell kinetic parameters measuring the promotional effect of the various compounds were estimated. On the basis of these estimates, we computed the number of nonextinct altered foci and their volume fraction as functions of time. We found that foci exhibiting the coextensive phenotype respond most efficiently to promoter treatment, while GGT dominant foci respond weakly to all the promoters with the exception of 3-MC. For phenobarbital, we observed a significant slowing of focal cell proliferation over time.

Adenosine Triphosphatases↗
PubMed1991

Effect of varying the concentration of phenobarbital and its duration of treatment on the evolution of carcinogen induced enzyme-altered foci in rat liver.

The present study was aimed to investigate whether the promoting activity of phenobarbital in rodent liver is related to its daily dose level and duration of treatment or rather to its total dose administered. For this purpose groups of female Wistar rats were treated for 5 consecutive days with an initiating dose of 10 mg/kg body weight N-nitrosodiethylamine. Subsequently, rats were given phenobarbital-sodium (PB) in their drinking water at concentrations of 20, 50, 100 and 200 mg/l for varying lengths of time, such that the total dose of xenobiotic was very similar throughout the different treatment groups ranging from approximately 950 to 1100 mg/kg body weight. The number and volume fraction of lesions negative for the marker enzyme adenosine triphosphatase in liver were subsequently scored as a means to determine the carcinogenic response in this organ. Slight promoting effects of PB were only seen at the lowest concentration of 20 mg/l, whereas no significant effects were observed at 50 and 100 mg/l. At the highest concentration of 200 mg/l an inhibition of carcinogenic response was obtained. Although the effects seen in this study were only moderate, our data favour the idea that the promoting effects of PB depend on the actual concentration of the compound and the duration of treatment rather than on the total dose administered.

Adenosine Triphosphatases↗