Early Stages Of Chemical Carcinogenesis Research Articles

Modulation of radical pairs dynamics immersed in an ELF-EMF: The effect on hepatocarcinogenesis

The most suitable mechanism of action of electromagnetic fields (EMF) on biological systems is the effect on the radical pair (RP) recombination through the Zeeman effect and hyperfine interaction, which changes the rate of reactions or the product distribution. Enzyme reactions with RP intermediates can be altered by EMF, like those catalyzed by cytochrome P450 enzymes (CYP450), a heme-thiolate family protein that detoxifies xenobiotics and involved in chemical carcinogenesis. CYP450 activate chemical carcinogens producing an enormous amount of free radicals, which damage the DNA resulting in the malignant transformation of cells. During the activation, CYP450 produce spin-correlated RP intermediates that can either go to recombination or to continue the catalytic process. As CYP450 are electron carrier proteins, it is possible that RP intermediates may be affected by EMF. It was previously found that periodic treatment with extremely low frequency electromagnetic fields (ELF-EMF) inhibits more than 50% the number and area of preneoplastic lesions in rats with chemically induced hepatocarcinogenesis through reduction of cell proliferation. In this work, we developed a quantum mechanical model based on RP mechanism in order to explain the experimental effects of ELF-EMF on the free radicals produced in the early stages of chemical carcinogenesis.

Genetic alteration of stemness factors and p53 in mouse forestomach by chemical carcinogen-induced carcinogenesis

The expression profiles of genes normally enriched in embryonic stem (ES) cells (stemness factors) are associated with poor clinical outcome in solid tumors. However, whether such gene expression is responsible for tumor initiation and progression remains to be determined. The tumor suppressor gene p53 is known to attenuate the expression of Nanog, which is essential for maintaining stem cells in response to DNA damage. On the basis of these findings, we hypothesized that stemness factors and p53 closely correlate with each other and form a network in response to genomic damage in the early phase of carcinogenesis and in the process of tumor progression. In this study, we applied the N-methylbenzylnitrosoamine (NMBA)-induced carcinogenesis model to the mouse forestomach to clarify the role of the stemness factors, c-Myc, Klf4, Sox2, Oct3/4 and Nanog, in cancer development using p53(+/+) (n=26) and p53(+/-) (n=11) C57BL/6J mice. Thirty weeks following NMBA administration, histologically evident squamous cell carcinoma was detected in the forestomachs of p53(+/+) mice, and the percentage of p53-positive nuclei in the forestomach epithelium gradually increased during carcinogenesis. Tumor development in p53(+/-) mice occurred significantly earlier than in p53(+/+) mice. Quantitative real-time PCR analyses revealed a reduced c-Myc and Klf4 expression before evident morphological changes were observed, and an increased expression with the development of squamous cell carcinoma. Sox2 expression remained unchanged until tumor development and increased with the appearance of squamous cell carcinomas. The expression of Oct3/4 and Nanog increased at the early stages following NMBA administration, and Nanog expression in situ was not positively affected by the deficiency of p53. Findings of the present study suggested that Oct3/4 may be involved in the progression of carcinogenesis from normal epithelial cells at early stages, suggesting the potential use of Oct3/4 as a biomarker in forestomach tumor formation at early stages of chemical carcinogenesis.

Arsenic Carcinogenesis in the Skin

Chronic arsenic poisoning is a world public health issue. Long-term exposure to inorganic arsenic (As) from drinking water has been documented to induce cancers in lung, urinary bladder, kidney, liver and skin in a dose-response relationship. Oxidative stress, chromosomal abnormality and altered growth factors are possible modes of action in arsenic carcinogenesis. Arsenic tends to accumulate in the skin. Skin hyperpigmentation and hyperkeratosis have long been known to be the hallmark signs of chronic As exposure. There are significant associations between these dermatological lesions and risk of skin cancer. The most common arsenic-induced skin cancers are Bowen's disease (carcinoma in situ), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Arsenic-induced Bowen's disease (As-BD) is able to transform into invasive BCC and SCC. Individuals with As-BD are considered for more aggressive cancer screening in the lung and urinary bladder. As-BD provides an excellent model for studying the early stages of chemical carcinogenesis in human beings. Arsenic exposure is associated with G2/M cell cycle arrest and DNA aneuploidy in both cultured keratinocytes and As-BD lesions. These cellular abnormalities relate to the p53 dysfunction induced by arsenic. The characteristic clinical figures of arsenic-induced skin cancer are: (i) occurrence on sun-protected areas of the body; (ii) multiple and recrudescent lesions. Both As and UVB are able to induce skin cancer. Arsenic treatment enhances the cytotoxicity, mutagenicity and clastogenicity of UV in mammalian cells. Both As and UVB induce apoptosis in keratinocytes by caspase-9 and caspase-8 signaling, respectively. Combined UVB and As treatments resulted in the antiproliferative and proapoptotic effects by stimulating both caspase pathways in the keratinocytes. UVB irradiation inhibited mutant p53 and ki-67 expression, as well as increased in the number of apoptotic cells in As-BD lesions which resulted in an inhibitory effect on proliferation. As-UVB interaction provides a reasonable explanation for the rare occurrences of arsenical cancer in the sun-exposed skin. The multiple and recurrent skin lesions are associated with cellular immune dysfunction in chronic arsenism. A decrease in peripheral CD4+ cells was noticed in the inhabitants of arsenic exposure areas. There was a decrease in the number of Langerhans cells in As-BD lesion which results in an impaired immune function on the lesional sites. Since CD4+ cells are the target cell affected by As, the interaction between CD4+ cells and epidermal keratinocytes under As affection might be closely linked to the pathogenesis of multiple occurrence of arsenic-induced skin cancer. In this review, we provide and discuss the pathomechanisms of arsenic skin cancer and the relationship to its characteristic figures. Such information is critical for understanding the molecular mechanism for arsenic carcinogenesis in other internal organs.

Cloning and Expression of the Catalytic Domain from Rat Hepatoma H35 Cell GDP-Fucose:GM1α1→2Fucosyltransferase, an Enzyme Which Is Activated during Early Stages of Chemical Carcinogenesis in Rat Liver

A ganglioside GM1-specific α1→2fucosyltransferase is induced during the early stages of chemical carcinogenesis withN-2-acetylaminofluorene (AAF) in rat liver hepatocytes. The induction of this enzyme gives rise to the expression of a fucose-containing ganglioside with the same determinant structure as blood group B on a GM1ganglioside core. Fucoganglioside synthesis is not found in normal rat liver but is elevated in premalignant liver and is often highly expressed in derived rat hepatoma cell lines. Based upon the consensus sequence from portions of previously cloned human, rabbit, and rat α1→2fucosyltransferase enzymes, primers were designed which were used in RT-PCR experiments with rat hepatoma H35 cell total RNA to generate cDNAs encoding the extracellular, catalytic domain of the H35 cell α1→2fucosyltransferase. Sequencing of these PCR fragments showed them to encode a novel enzyme with high homology to other cloned enzymes, particularly secretor α1→2fucosyltransferases. The derived sequence indicated that the 3′ portion of the gene was virtually identical to the α1→2fucosyltransferase B (FTB) fragment reported earlier in rat PROb colon-adenocarcinoma cells (J-P. Piauet al.Biochem. J. 300, 623–626, 1994). A PCR product corresponding to the H35 cell α1→2fucosyltransferase was obtained from total RNA isolated from F344 rat liver after 0.03%N-2-acetylaminofluorene administration. No PCR product was obtained from total RNA isolated from normal F344 liver using PCR primers for the H35 cell α1→2fucosyltransferase. The H35 cell α1→2fucosyltransferase was expressed in the pPROTA vector and the derived fusion protein demonstrated the ability to transfer fucose to ganglioside GM1but not to the neolacto-series acceptor nLcOse4Cer.

Characterization of sodium and chloride conductances in preneoplastic and neoplastic murine colonocytes.

Glucocorticoids, such as dexamethasone, induce amiloride-sensitive Na+ conductances in rat distal colon epithelium. The activity of these conductances diminishes from the surface to the base of the crypt whereas cAMP-stimulated Cl- secretion decreases from the crypt base to the surface. These gradients are likely to be perturbed during carcinogenesis. We therefore determined the magnitude of Na+ and Cl- conductances in colonocytes isolated from normal and carcinogen-treated rats. Colon carcinogenesis was induced by injection of dimethylhydrazine (DMH) (18 mg/kg) for 5 weeks. Before sacrifice animals were treated for 3 days with dexamethasone. Colonocyte populations from the surface to the crypt base (C1-C5) were harvested from the distal colon by a Ca2+-chelating procedure. The activity of Na+ conductances was determined by uptake of 22Na+ by surface and crypt colonocyte populations and by membrane vesicles in the presence and absence of 10 microM amiloride. In control rats Na+ conductance was highest in surface colonocytes and absent in the crypt base. As early as 2 weeks after initiation of DMH treatment amiloride-inhibited Na+ uptake was virtually absent in the upper crypt. Transcriptional assessment of the alpha-, beta- and gamma-subunits that constitute the epithelial Na+ channel revealed that DMH treatment reduces the expression of beta-subunit mRNA. We then examined 36Cl- efflux from isolated colonocytes of normal and carcinogen-treated rats in response to forskolin (0.01 mM). Forskolin induced a marked rise in cAMP in lower crypt cells concomitant with a significant stimulation of 36Cl- efflux. Intracellular cAMP increased in upper crypt cells in response to forskolin without an increase in 36Cl- efflux. By contrast, upper crypt colonocytes from DMH-treated rats showed forskolin-stimulated efflux beginning 4 weeks after initiation of treatment. We conclude that induction of Na+ conductances by glucocorticoids is inhibited during the early stages of chemical carcinogenesis due to lack of induction of the beta-subunit of the channel. By contrast, Cl- transport is stimulated both in surface and lower crypt cell compartments during different stages of chemical carcinogenesis.

Selective colocalization of lipid peroxidation and protein thiol loss in chemically induced hepatic preneoplastic lesions: the role of gamma-glutamyltranspeptidase activity.

A number of studies indicate that cell proliferation can be modulated by changes in the redox balance of (soluble and protein) cellular thiols. Free radical processes, including lipid peroxidation (LPO), can affect such a balance, and a role for LPO in multistage carcinogenesis has been envisaged. The present study was aimed to assess the relationships between the protein thiol redox status and the LPO process in chemically induced preneoplastic tissue. The Solt-Farber's initiation-promotion model of chemical carcinogenesis in the rat liver was used. In fresh cryostat sections, preneoplastic lesions were identified by the reexpression of gamma-glutamyltranspeptidase (GGT) activity. In serial sections, different classes of protein thiols were stained; in additional sections, LPO was elicited by various prooxidant mixtures and determined thereafter by the hydroxynaphthoic hydrazide-Fast Blue B procedure. The incubation of sections in the presence of chelated iron plus substrates for GGT activity leads to the development of LPO in selected section areas closely corresponding to GGT-positive lesions, indicating the ability of GGT activity to initiate LPO. Protein-reactive thiols, as well as total protein sulfur, were decreased by 20-25% in cells belonging to GGT-positive preneoplastic nodules, suggesting the occurrence of oxidative conditions in vivo. The incubation of additional adjacent sections with the prooxidant mixture H2O2 plus iron(II), in order to induce the complete oxidation of lipid present in the section, showed a decreased basal concentration of oxidizable lipid substrate in GGT-rich areas. The decreased levels of both protein thiols and lipid-oxidizable substrate in GGT-positive nodules suggest that the observed GGT-dependent pathway of LPO initiation can be chronically operative in vivo during early stages of chemical carcinogenesis, in cells expressing GGT as part of their transformed phenotype.

Expression of hepatitis B surface and core antigens and transforming growth factor-alpha in "oval cells" of the liver in patients with hepatocellular carcinoma.

Recent studies have identified epithelial cell populations in human livers that are similar to the "oval cells" and "transitional cells" seen in rat livers during the early stages of chemical carcinogenesis. It has been suggested that these cells might be precursors of hepatocytes and theoretically could be involved in hepatocarcinogenesis. The hepatitis B virus (HBV) also is believed to play a role in the etiology of hepatocellular carcinoma (HCC). Therefore, a study was conducted in nontumorous livers adjacent to HCCs obtained from 26 patients from China to determine whether HBV antigens could be identified in oval cells and transitional cells using an immunohistochemical technique. Hepatitis B surface antigen (HBsAg) was detected in the nontumorous livers of 22/26 (85%) patients. HBsAg was detected in oval cells in 18/26 (69%), in transitional cells in 21/26 (81%), and in mature hepatocytes in 22/26 (85%), but not in bile duct or ductule cells. Transforming growth factor-alpha (TGF-alpha) was expressed in oval cells, transitional cells, and bile duct cells in 24/26 (92%) patients, an in mature hepatocytes in 25/26 (96%). Coexpression of HBsAg and TGF-alpha was identified in the same cells in populations of oval cells and transitional cells of selected patients. Because of the possibility that oval cells could be a source of evolving HCC, these findings suggest that expression of TGF-alpha associated with HBV infection of oval cells could be a mechanism of human hepatocarcinogenesis. Thus, oval cells could be a site (or one of the sites) where HBV participates in the development of HCC.

Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver.

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.

Minimal neoplasias in experimental liver carcinogenesis.

The rodent liver provides a unique opportunity to study the early stages of chemical carcinogenesis. If a rat or a mouse is exposed for a long period to a hepatocarcinogen, hepatocellular carcinomas develop which invade and replace part of the normal liver, and sometimes tumor cells disseminate to give rise to lung metastasis. Long before this final stage is reached cellular changes can be observed in the liver parenchyma. Foci of altered hepatocytes appear which show an aberration from the enzymatic pattern of normal hepatocytes. These foci can best be demonstrated in enzyme histochemical preparations in cryostat sections prepared from the liver at early stages of carcinogen exposure. Small islands of hepatocytes are then seen which show a deficiency of glucose-6-phosphatase as described in a preliminary report first in 1964 by the Friedrich-Freksa group (Gossner and Friedrich-Freksa 1964). In the same year, Bannasch and Muller reported that during hepatocarcinogenesis foci of glycogen-storing cells can be observed and they related their occurrence to the previous exposure to a hepatocarcinogen. Interestingly, it was in 1961 that Grundmann pointed out that basophilic foci occur in the liver after carcinogen exposure and he suggested that these subpopulations in the liver might represent precursor lesions in hepatocarcinogenesis.

Surface ultrastructural changes in chemically induced premalignant lesions in the hamster cheek pouch.

The histopathological and topographical alterations occurring during the early stages of chemical carcinogenesis in the hamster cheek pouch using 7,12-dimethylbenz (alpha) anthracene (DMBA) were studied by light and scanning electron microscopy. At the scanning electron microscopic level, cellular overlapping and mild disturbance of intercellular bridges were noticed as early as the second week of DMBA application; these changes progressed with time. Clear epithelial dysplastic changes at the light microscopic level were detected by the sixth week of the experiment. The results of this investigation demonstrate the usefulness of scanning electron microscopy as an adjunct tool to study early alterations in cell morphology which occur in the stratified squamous epithelium of the hamster cheek pounch in response to a chemical carcinogen.

Cell proliferation and DNA repair in the liver during early stages of chemical carcinogenesis

X-chromosomal phosphoglycerate kinase mocaicisms in organ samples of female heterozygous mice provided a means to prove, because of a selective expression of one of the two allozymes, the clonal origin of carcinogen-induced preneoplastic hepatocellular populations (see also ref. 61). The occurrence of these clonal preneoplastic foci was used in rats to determine cell cycle dependent differences of transformation sensitivity and DNA repair. The highest transformation rate was found after carcinogen exposure in late G 1/early S phase of the cell cycle. Experimental disturbance of DNA precursor pools by continuous thymidine infusion during carcinogen exposure results in an increased formation of preneoplastic clones. This is a further argument in favor of an essential role of base-mispairing during initiation. Cell cycle dependent fluctuations of O 6-methylguanine DNA transferase with an increasing enzyme activity in late G 1 and a maximum in early S phase indicate that cells possess an increased potential for eliminating promutagenic O 6-methylguanine during the most transformation sensitive parts of the cell cycle to prevent base-mispairing during DNA replication or transcription. However, this putatively protective effect is limited because the enzyme is rapidly expended in the reaction and drops again in later stages of the cell cycle.

Cellular basis for the loss of carcinogen from methylcholanthrene-impregnated Millipore membrane

Millipore 'discs' impregnated with methylcholanthrene dissolved in wax or in crystalline form implanted subcutaneously in mice evoke an early intense macrophage and giant cell reaction; later the discs become covered with connective tissue, and eventually fibrosarcomas develop in their vicinity. Studies with tritium-labelled material show that under these conditions methylcholanthrene (MC) is removed from the discs and broken down rapidly (half life about 7 days) to a water-soluble product, which diffuses locally, is distributed widely via the blood stream, and is excreted in faeces and urine. Removal of label is halted by whole-body irradiation (550 R) with the disc area shielded; this observation, in conjunction with the histological and autoradiographic findings and the paucity of label in cells stripped from excised discs, points to the conclusion that the removal of MC from impregnated discs, and its subsequent degradation, depend on the presence of macrophages and the continual replacement of spent macrophages by new cells generated centrally. The rate of disappearance of label from implanted [3H]MC discs was not altered by administration of Corynebacterium parvum; this, however, does not exclude the possibility that the metabolic pathways involved in the removal of MC are altered. To investigate this it is proposed to study, in both normal mice and mice treated with C. parvum, the extent to which cytochrome P450 and other enzymes concerned in the activation and detoxification of polycyclic hydrocarbons by liver microsomal fractions are inducible in the macrophages that accumulate in inflammatory exudates. This histological techniques used should be readily applicable to the study of the early stages of chemical carcinogenesis and the host reaction to transformed cells.

Inhibition by 5-fluorouracil of the early stages of chemical carcinogenesis in mouse skin.

Summary5-FU was found to inhibit the early stages of DMBA-induced epidermal carcinogenesis in the mouse. The dose which must be employed to obtain this effect is of the same order of magnitude as that required for bringing about regression of established tumors of the mouse skin.

Nuclear RNA synthesis in rat liver during the early stages of chemical carcinogenesis.

Nuclear RNA synthesis in rat liver during the early stages of chemical carcinogenesis.