Biomedical subjects

S J James

Publications and source records attributed to S J James.

At least 37 records · Page 2Linked to original sources

PubMed1999

Non-uniformity of surface temperatures after microwave heating of poultry meat.

Microwave energy has the potential to raise the surface temperatures of meat rapidly for a short period of time sufficient to reduce bacterial numbers significantly without causing physical changes to the meat. Studies have investigated the ability of a standard domestic microwave oven (2450 MHz; IEC 1191 W), an experimental repeatable microwave cavity (2450 MHz; IEC 1139 W) and a number of shielding techniques to achieve uniform surface temperature distributions on pieces of poultry meat. In the domestic oven temperature differences of up to 60 and 80 degrees C were found between different points on the surface of the same sample after 30 s and 3 minutes of heating respectively. The use of a standard cavity and shielding resulted in a difference of less than 5 degrees C between the average surface temperature on the edge and middle of regular slabs of chicken after 30 s exposure. Results show that microwave heating, using 2450 MHz, is unlikely to produce consistently uniform enough surface temperatures on meat to reduce bacterial numbers without surface damage.

Animals↗

PubMed1999

Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome.

BACKGROUND: Down syndrome, or trisomy 21, is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. The origin of the extra chromosome is maternal in 95% of cases and is due to the failure of normal chromosomal segregation during meiosis. Although advanced maternal age is a major risk factor for trisomy 21, most children with Down syndrome are born to mothers <30 y of age. OBJECTIVE: On the basis of evidence that abnormal folate and methyl metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation, we hypothesized that the C-to-T substitution at nucleotide 677 (677C-->T) mutation of the methylenetetrahydrofolate reductase (MTHFR) gene may be a risk factor for maternal meiotic nondisjunction and Down syndrome in young mothers. DESIGN: The frequency of the MTHFR 677C-->T mutation was evaluated in 57 mothers of children with Down syndrome and in 50 age-matched control mothers. Ratios of plasma homocysteine to methionine and lymphocyte methotrexate cytotoxicity were measured as indicators of functional folate status. RESULTS: A significant increase in plasma homocysteine concentrations and lymphocyte methotrexate cytotoxicity was observed in the mothers of children with Down syndrome, consistent with abnormal folate and methyl metabolism. Mothers with the 677C-->T mutation had a 2.6-fold higher risk of having a child with Down syndrome than did mothers without the T substitution (odds ratio: 2.6; 95% CI: 1.2, 5.8; P < 0.03). CONCLUSION: The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene.

Adult↗

PubMed1998

Hypomethylation of the rat glutathione S-transferase pi (GSTP) promoter region isolated from methyl-deficient livers and GSTP-positive liver neoplasms.

DNA methylation at the 5-position on the cytosine ring in CpG dinucleotides (CpG sites) appears to play an important role in regulating gene expression. In general, there is an inverse relationship between promoter CpG site methylation and the potential for transcription. Thus, changes in DNA methylation density may lead to altered levels of proteins such as glutathione S-transferase pi (GSTP), which is frequently used as a marker to detect hepatocellular foci and neoplasms in the rat. In the present study, the level of CpG methylation in the rat GSTP promoter region was determined in bisulfite-treated DNA isolated from control (untreated) rat livers, chemically induced, GSTP-positive rat liver neoplasms, and methyl-deficient rat livers that contained numerous GSTP-positive foci after administration of a defined diet deficient in folate and choline and low in methionine (0.18%). Eight cytosines between -235 and + 140 in the GSTP promoter region were methylated in a site-specific manner in GSTP-negative control liver, whereas these same sites were hypomethylated in all four chemically-induced, GSTP-positive neoplasms. Similarly, all CpG sites were unmethylated in methyl-deficient liver DNA within 3 weeks of the rats receiving the methyl-deficient diet, and they remained unmethylated throughout the 36-week treatment period. Five of the eight CpG sites are located within consensus sequences for the DNA binding proteins Spl and E2F. This indicates at least one possible mechanism that could potentially lead to transcriptional activation of GSTP in hepatocellular foci and neoplasms during rat hepatocarcinogenesis. These findings suggest that methylation of critical cytosines within the promoter region rather than all CpG-associated cytosines may be a determining factor in regulation of GSTP expression.

Animals↗

PubMed1998

Moderate folate depletion increases plasma homocysteine and decreases lymphocyte DNA methylation in postmenopausal women.

To determine the human folate requirement on the basis of changes in biochemical pathways, we studied the effect of controlled folate intakes on plasma homocysteine and lymphocyte DNA methylation and deoxynucleotide content in healthy postmenopausal women. Eight women (49-63 y of age) were housed in a metabolic unit and fed a low folate diet containing 56 microg/d of folate for 91 d. Folate intake was varied by supplementing 55-460 microg/d of folic acid (pteroylglutamic acid) to the diet to provide total folate intake periods of 5 wk at 56 microg/d, 4 wk at 111 microg/d and 3 wk at 286-516 microg/d. A subclinical folate deficiency with decreased plasma folate was created during the first two periods. This resulted in significantly elevated plasma homocysteine and urinary malondialdehyde, and lymphocyte DNA hypomethylation. The folate depletion also resulted in an increased ratio of dUTP/dTTP in mitogen-stimulated lymphocyte DNA and decreased lymphocyte NAD, changes suggesting misincorporation of uracil into DNA and increased DNA repair activity. The DNA hypomethylation was reversed with 286-516 microg/d of folate repletion, whereas the elevated homocysteine decreased with 516 but not 286 microg/d of folate. The results indicate that marginal folate deficiency may alter DNA composition and that the current RDA of 180 microg/d may not be sufficient to maintain low plasma homocysteine concentrations of some postmenopausal women.

Creatinine↗

PubMed1998

Fetal brain death syndrome--a case report and literature review.

This case describes a new feature of fetal brain death syndrome, abnormal movements mimicking fetal convulsions being subsequently found to be decerebrate hypertonicity in a brain-dead fetus. It also confirms the diagnostic criteria of fetal brain death, both clinical and ultrasonic. The development of polyhydramnios both prior to and after the presumed neurological event is suggested as an association with the diagnosis of fetal brain death. Increased awareness of this event and the heterogeneity of the presentation may prevent further unnecessary Caesarean sections, as to date only 4 of the 10 cases in the literature were diagnosed prenatally. Utilization of techniques such as fetal blood sampling should be considered to further delineate the diagnosis.

Adult↗

PubMed1998

Upregulation of apoptosis with dietary restriction: implications for carcinogenesis and aging.

The maintenance of cell number homeostasis in normal tissues reflects a highly regulated balance between the rates of cell proliferation and cell death. Under pathologic conditions such as exposure to cytotoxic, genotoxic, or nongenotoxic agents, an imbalance in these rates may indicate subsequent risk of carcinogenesis. Apoptotic cell death, as opposed to necrotic cell death, provides a protective mechanism by selective elimination of senescent, preneoplastic, or superfluous cells that could negatively affect normal function and/or promote cell transformation. The relative efficiency or dysfunction of the cell death program could therefore have a direct impact on the risk of degenerative or neoplastic disease. Dietary restriction of rodents is a noninvasive intervention that has been reproducibly shown to retard tumor development and most physiologic indices of aging relative to ad libitum-fed animals. As such, it provides a powerful model in which to study common mechanistic processes associated with both aging and cancer. In a recent study we established that chronic dietary restriction (DR) induces an increase in spontaneous apoptotic rate and a decrease in cell proliferation rate in hepatocytes of 12-month-old B6C3F1 DR mice relative to ad libitum (AL)-fed mice. This diet-induced shift in cell death/proliferation rates was associated with a marked reduction in subsequent development of spontaneous hepatoma and a marked increase in disease-free life span in DR relative to AL-fed mice. These results suggest that total caloric intake may modulate the rates of cell death and proliferation in a direction consistent with a cancer-protective effect in DR mice and a cancer-promoting effect in AL mice. To determine whether the increase in spontaneous apoptotic rate was maintained over the life span of DR mice, apoptotic rates were quantified in 12-, 18-, 24- and 30-month-old DR and AL mice. The rate of apoptosis was elevated with age in both diet groups; however, the rate of apoptosis was significantly and consistently higher in DR mice regardless of age. In double-labeling experiments, an age-associated increase in the glutathione S-transferase-II expression in putative preneoplastic hepatocytes in AL mice was rapidly reduced by apoptosis upon initiation of DR. Thus, intervention that promote a low-level increase in apoptotic cell death may be expected to protect genotypic and phenotypic stability with age. If during tumor promotion an adaptive increase in apoptosis effectively balances the dysregulated increase proliferation, the risk of permanent genetic error and carcinogenesis would be minimized.

Aging↗

PubMed1997

Alterations in hepatic p53 gene methylation patterns during tumor progression with folate/methyl deficiency in the rat.

Chronic dietary methyl deficiency in F344 rats was used as an in vivo mammalian model in which to evaluate the gene-specific alterations in DNA methylation patterns during multistage hepatocarcinogenesis. Using bisulfite mapping, the site-specific methylation profile within exons 6-7 of the 53 gene was determined in control liver, preneoplastic nodules (after 36 weeks of folate/methyl deficiency) and in hepatocellular carcinoma (after 54 weeks of deficiency). A progressive loss of methyl groups was observed at most CpG sites on both coding and non-coding strands during the first 36 weeks of folate/methyl deficiency, with the greatest loss occurring on the coding strand. When the same sequence was evaluated in tumor DNA after 54 weeks of deficiency, the majority of cytosines were unexpectedly found to have become remethylated. CpG sites that had previously lost methyl groups on both strands during preneoplasia as well as CpG sites that had been constitutively non-methylated, had undergone de novo methylation in tumor DNA. Maintenance methyltransferase and de novo methyltransferase activity in nuclear extracts were assessed using hemimethylated and non-methylated DNA substrates, respectively. In tumor, de novo methyltransferase capacity was increased approximately 4-fold relative to control or preneoplastic liver and associated with a relative increase in both p53 and genome-wide methylation density. In the preneoplastic nodules, the level p53 mRNA was increased and associated with hypomethylation in the coding region of the gene, whereas in tumor tissue, p53 mRNA was decreased and associated with relative hypermethylation. Taken together, these results provide additional insights into the dysregulation and instability in DNA methylation that accompanies the transition to tumor.

Animals↗

PubMed1997

P53 synthesis and phosphorylation in the aging diet-restricted rat following retinoic acid administration.

Multiple doses of retinoic acid (RA) were administered intraperitoneally to three groups of male Fischer 344 rats over a 36 h period. The p53 isoforms from bone marrow nuclei in these three groups of rats were analyzed over time by two-dimensional polyacrylamide gel electrophoresis (PAGE) and fluorography for the incorporation of [35S]methionine (p53-synthesis) and [32P]phosphate (p53-phosphorylation). Two groups of rats, young (3.5 months) ad libitum (Y/AL) and old (28 months) ad libitum (O/AL), had free access to Purina rat chow; a third group of old (28 months) diet-restricted rats (O/DR) were maintained on a restricted caloric intake (60% of the AL diet) from 3 months of age. After 36 h of RA dosing, the PAGE patterns of p53 synthesis and phosphorylation in Y/AL and O/DR rats were very similar. In both groups, an increase in complexity was observed with labeling of additional isotypes possessing more acidic isoelectric values. In contrast, the O/AL animals showed a pattern of p53 isoform synthesis and phosphorylation that was considerably less complex and lacked the pronounced shift to more acidic forms following RA dosing. The p53 isoforms of O/AL rats as recognized by wild type (wt) Pab 246 antibody, were also much less dramatic in their increase to more acidic forms. Two-dimensional phospho-tryptic maps of Y/AL and O/DR rats were also very similar, both exhibiting two additional minor 32P-labeled fragments after RA dosing. The maps of O/AL rats did not show the two additional fragments following RA administration. After RA dosing, cyclin protein inhibitors (p16, p21, p27) revealed robust labeling with their respective antibodies in Y/AL and O/DR rats as analyzed by Western blotting. The O/AL animals showed marginally detectable antibody recognition of the cyclin inhibitors after RA dosing. Taken together, these data suggest that the biosynthesis and phosphorylation of p53 isoforms and the expression of cyclin dependent kinase inhibitor proteins is not significantly different between Y/AL and O/DR rats. Further, these results confirm and extend our previous observations that chronic diet-restriction attenuates the age related decline in the metabolic activity of nuclear protein products.

Aging↗

PubMed1997

Characterization of cellular response to silicone implants in rats: implications for foreign-body carcinogenesis.

Foreign-body (FB) carcinogenesis is a classic model of multistage tumour development in rodents. Previous studies have demonstrated that the physical characteristics of the implant, and not the chemical composition, are the critical determinants of tumour development. The recent controversy over silicone breast implants has raised questions regarding the potential carcinogenicity of lifetime tissue exposure to silicone products. The present study was designed to determine whether the inflammatory and fibrotic reactions associated with silicone implants are due to a non-specific foreign-body reaction or whether these responses reflect the unique chemical composition of silicone. F344 rats were implanted subcutaneously with one of three biomaterials: silicone elastomer (Group 1); impermeable cellulose acetate filters (Group 2, positive control); or porous cellulose acetate filters (Group 3, negative control). The silicone and cellulose implants of Groups 1 and 2 have been previously shown to induce fibrosarcomas in rodents, whereas the porous cellulose acetate implants of Group 3 have been shown to be non-carcinogenic. One week and two months after implantation, the pericapsular tissues were evaluated using histopathological and in situ immunohistochemical analyses. Endpoints included expression of leucocyte antigens CD4 (T helper/inducer), CD8 (T suppressor/cytotoxic) and CD11 b/c (macrophage), proliferating cell nuclear antigen (PCNA) as an indicator of proliferation, and in situ end-labelling (ISEL) of 3'OH DNA strand breaks as an indicator of DNA damage and apoptosis. The results indicated that the acute and chronic cellular responses to silicone (Group 1) were not different from impermeable cellulose filters (Group 2) of identical size and shape, suggesting that these responses were not unique to silicone. The inflammatory response to the carcinogenic cellulose and silicone implants (Groups 1 and 2) was attenuated and associated with the formation of a thick fibrotic capsule. In contrast, the porous cellulose filters (Group 3) induced a markedly different cellular response in which the inflammatory reaction was more extensive, prolonged and associated with minimal fibrosis. Within the fibrotic capsule surrounding the tumorigenic implants, but not the non-tumorigenic implants, cell proliferation and apoptotic cell death were increased and associated with persistent DNA strand breaks. Taken together, the results suggest that the micrometre-scale surface morphology of the implant determines the nature of the subsequent cellular response which may predispose to tumour development. Further, these studies serve to emphasize the critical importance of appropriate physical controls in studies designed to evaluate carcinogenic or autoimmune manifestations associated with silicone implants in order to rule out the contribution of the chronic foreign-body reaction.

Acute Disease↗

PubMed1997

Folate deficiency in rats induces DNA strand breaks and hypomethylation within the p53 tumor suppressor gene.

Folate is essential for the de novo biosynthesis of purines and thymidylate, and is an important mediator in the transfer of methyl groups for DNA methylation. Folate deficiency, therefore, could contribute to abnormal DNA integrity and methylation patterns. We investigated the effect of isolated folate deficiency in rats on DNA methylation and DNA strand breaks both at the genomic level and within specific sequences of the p53 tumor suppressor gene. Our data indicate that folate deficiency induces DNA strand breaks and hypomethylation within the p53 gene. Such alterations either did not occur or were chronologically delayed when examined on a genome-wide basis, indicating some selectivity for the exons examined within the p53 gene. Folate insufficiency has been implicated in the development of several human and experimental cancers, and aberrations within these regions of the p53 gene that were examined in this study are thought to play an integral role in carcinogenesis. The aforementioned molecular alterations may therefore be a means by which dietary folate deficiency enhances carcinogenesis.

Animals↗

PubMed1997

Presence and consequence of uracil in preneoplastic DNA from folate/methyl-deficient rats.

Uracil can arise in DNA by misincorporation of dUTP into nascent DNA and/or by cytosine deamination in established DNA. Based on recent findings, both pathways appear to be promoted in the methyl-deficient model of hepatocarcinogenesis. A chronic increase in the ratio dUTP:dTTP with folate/methyl deficiency can result in a futile cycle of excision and reiterative uracil misincorporation leading to premutagenic apyrimidinic (AP) sites, DNA strand breaks, DNA fragmentation and apoptotic cell death. The progressive accumulation of unmethylated cytosines with chronic methyl deficiency will increase the potential for cytosine deamination to uracil and further stress uracil mismatch repair mechanisms. Uracil is removed by a highly specific uracil-DNA glycosylase (UDG) leaving an AP site that is subsequently repaired by sequential action of AP endonuclease, 5'-phosphodiesterase, a DNA polymerase and DNA ligase. Since the DNA polymerases cannot distinguish between dUTP and dTTP, an increase in dUTP:dTTP ratio will promote uracil misincorporation during both DNA replication and repair synthesis. The misincorporation of uracil for thymine (5-methyluracil) may constitute a genetically significant form of DNA hypomethylation distinct from cytosine hypomethylation. In the present study a significant increase in the level of uracil in liver DNA as early as 3 weeks after initiation of folate/methyl deficiency was accompanied by parallel increases in DNA strand breaks, AP sites and increased levels of AP endonuclease mRNA. In addition, uracil was also detected within the p53 gene sequence using UDG PCR techniques. Increased levels of uracil in DNA implies that the capacity for uracil base excision repair is exceeded with chronic folate/methyl deficiency. It is possible that enzyme-induced extrahelical bases, AP sites and DNA strand breaks interact to negatively affect the stability of the DNA helix and stress the structural limits of permissible uracil base excision repair activity. Thus substitution of uracil for thymine induces repair-related premutagenic lesions and a novel form of DNA hypomethylation that may relate to tumor promotion in the methyl-deficient model of hepatocarcinogenesis.

Animals↗

PubMed1997

Apoptosis and proliferation under conditions of deoxynucleotide pool imbalance in liver of folate/methyl deficient rats.

Weanling male F344 rats were fed either a semi-purified diet low in methionine and lacking in choline and folic acid (folate/methyl deficient) or a supplemented control diet for periods of 2, 5, 7 days, 3 weeks, and 9 weeks. Two days after initiating the folate/methyl deficient diet in weanling F344 rats, the incidence of apoptotic bodies, identified by in situ end-labeling of 3'-OH DNA strand breaks, was significantly increased in liver sections from the deficient rats. Apoptotic cell death was confirmed biochemically by an increase in nuclear Ca2+/Mg2+-dependent endonuclease activity that paralleled the increase in apoptotic bodies over the 9-week feeding period. There was no morphologic evidence of necrotic foci or necrosis-associated inflammatory response over the 9-week period. Confirming that cell turnover is chronically elevated in this model, the increase in apoptotic rate was accompanied by a sustained increase in the mitotic index (MI). The DNA repair-associated enzyme, poly(ADPribose) polymerase (PARP), was similarly elevated and was associated with significant decreases in the substrate for ADPribose polymer synthesis, nicotinamide adenine dinucleotide (NAD). Because folate metabolites are essential for de novo purine and thymidine biosynthesis, prolonged deficiency in folic acid can induce an imbalance in the deoxynucleotide precursors for DNA replication/repair and negatively affect the fidelity of DNA synthesis. Using an HPLC method, hepatic deoxyuridine triphosphate (dUTP) levels were increased at 3 and 9 weeks after initiation of the deficient diet and levels of thymidine triphosphate (dTTP) were reduced. An increase in dUTP/ dTTP ratio is consistent with a block in folate-dependent de novo thymidylate biosynthesis and may predispose to uracil misincorporation and DNA repair-related DNA strand breaks.

Animals↗

PubMed1997

Chemoprevention of spontaneous tumorigenesis in nullizygous p53-deficient mice by dehydroepiandrosterone and its analog 16alpha-fluoro-5-androsten-17-one.

Transgenic mice with both alleles of the p53 tumor suppressor gene product 'knocked out' by gene targeting are susceptible to early development of tumors, chiefly lymphomas and sarcomas. Compared with the control group, administration of dehydroepiandrosterone (DHEA) at 0.3% of the diet to male p53-deficient mice extended their lifespan by delaying death due to neoplasms (from 105 to 166 days on study, P = 0.002), primarily by suppressing lymphoblastic lymphoma (from 45 to 6% of neoplastic deaths, P = 0.010). Treatment with a synthetic DHEA analog, 16alpha-fluoro-5-androsten-17-one (compound 8354), at 0.15% of the diet also increased lifespan, to 140 days for mice that developed tumors (P = 0.037). The effects of these steroids on lifespan and tumor development did not appear to be strongly related to inhibition of food consumption and weight gain, in that a group pair-fed with control diet to the reduced food consumption of the DHEA-treated group developed and died of the same types of neoplasms at the same rate as the controls fed ad libitum. The chemopreventive effect of these steroids has been proposed to be due to suppression of DNA synthesis by inhibition of glucose 6-phosphate dehydrogenase, the rate-limiting enzyme of the pentose phosphate pathway. Although DHEA and its analog are strong non-competitive inhibitors of this enzyme in vitro, treatment with DHEA did not deplete cellular nucleotide pools in the liver, as would have been predicted. The chemopreventive effect of DHEA in this model may be due to steroid-induced thymic atrophy and suppression of T cell lymphoma, permitting these mice to survive long enough to develop tumors with longer latency.

Androstenes↗

PubMed1997

Dietary nucleotides: effects on cell proliferation following partial hepatectomy in rats fed NIH-31, AIN-76A, or folate/methyl-deficient diets.

The requirement of a number of tissues for dietary nucleotides could explain some of the differences observed in animals fed natural ingredient diets vs. those fed purified diets lacking a source of dietary nucleotides. Lack of dietary nucleotides is exacerbated in animals fed folate- or methyl-deficient semipurified diets, in which both salvage and folate-dependent de novo synthetic pathways are diminished. We examined hepatocyte proliferation following partial hepatectomy in weanling male Fischer-344 rats fed natural ingredient NIH-31 diet, nucleotide-free purified AIN-76A diet or a basal diet similar to AIN-76A but deficient in the methyl donors folate, choline and methionine. Additional groups were fed AIN-76A or folate/methyl-deficient diets supplemented with 0.25% yeast RNA. Compared with NIH-31, AIN-76A increased dUMP/dTTP ratios, reduced the mitotic index (MI) and increased the ratio of proliferating cell index (PCI) to mitotic cells, an indication that hepatocytes were delayed in S-phase. Addition of yeast RNA to AIN-76A reversed (by approximately 50%) the effects of AIN-76A on dUMP/dTTP and cell proliferation. A folate/methyl-deficient diet also produced an increased dUMP/dTTP ratio and markedly reduced the MI, increasing the PCI/MI, which suggested even further delay of cells in S-phase. Addition of yeast RNA to the folate/methyl-deficient diet was effective in significantly reversing the effects of folate/methyl deficiency.

Animal Feed↗

PubMed1996

DNA fragmentation and nuclear endonuclease activity in rat brain after severe closed head injury.

Previous studies have suggested that brain cells undergo apoptotic cell death during several neurodegenerative disorders such as Alzheimer's disease, Parkinsonism and ischemic stroke. In the present study, apoptotic DNA fragmentation and activation of nuclear endonuclease were evaluated in rat brain cells after head trauma. Severe closed head injury was induced in rats by the impact of a 450-g weight dropped from a height of 2 m. A 12% mortality was experienced after head trauma. Brain cell nuclei and DNA were isolated at intervals of 3, 10, 24 h, 3 and 10 days after head trauma. DNA fragmentation was measured by the random oligonucleotide-primed synthesis (ROPS) assay and was significantly increased with the maximum level of DNA fragmentation occurring at 10 h after trauma. The DNA and nuclei yields decreased 10 h after injury and remained at a reduced level at all subsequent sampling intervals. The DNA fragmentation induced after severe head trauma was accompanied by an increase in the activity of the Ca/Mg-dependent endonuclease associated with apoptosis. These data indicate that severe head injury is associated with significant brain cell death by apoptosis.

Animals↗

PubMed1996

Ethanol feeding of micropigs alters methionine metabolism and increases hepatocellular apoptosis and proliferation.

Chronic alcoholism is associated with increased cancer risk that may be related to ethanol-induced alterations in methionine and deoxynucleotide metabolism. These metabolic relationships were studied in micropigs fed diets for 12 months that contained 40% ethanol or cornstarch control with adequate folate. Ethanol feeding altered methionine metabolism without changing mean terminal liver folate levels. After initial equilibration to diet, ethanol feeding significantly increased monthly serum homocysteine levels while reducing serum methionine levels over the time course of the experiment. After 12 months, hepatic methionine synthase activity and the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) were significantly reduced in ethanol-fed animals, whereas the ratio of liver deoxyuridine triphosphate (dUTP) to deoxythymidine triphosphate (dTTP) was increased and correlated inversely with methionine synthase activity. These findings were associated with increased frequency of hepatocytes with apoptotic bodies and positivity for proliferating cell nuclear antigen (PCNA) in livers from ethanol-fed minipigs. These studies suggest that chronic ethanol feeding perturbs methionine metabolism by impairment of methionine synthase activity, resulting in deoxynucleoside triphosphate (dNTP) imbalance, increased apoptosis, and regenerative proliferation. These biochemical alterations may provide a promoting environment for carcinogenesis during long-term ethanol exposure.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1996

Quantification of 3'OH DNA breaks by random oligonucleotide-primed synthesis (ROPS) assay.

A simple and precise assay is presented for quantification of the relative number of 3'OH ends (breaks) present in DNA molecules. The assay is based on the ability of the Klenow fragment polymerase to initiate random oligonucleotide-primed synthesis from the reannealed 3'OH ends of single-stranded (ss) DNA. After a denaturation-reassociation step, the ssDNA serves as its own primer by randomly reassociating itself or to other ssDNA molecules. Under strictly defined reaction conditions (time, temperature, concentration of precursors) the incorporation of [32P]dNTP into newly synthesized DNA will be proportional to the initial number of 3'OH ends (breaks). The assay is specific for the detection of 3'OH ends and requires only 0.25 micrograms of DNA for analysis. It has application for the detection of the relative number of breaks per DNA molecule generated in vitro by endonucleases or in vivo during normal processes of DNA repair and also for the detection of DNA strand breaks from genotoxic DNA damaging agents. Although specific for 3'OH DNA ends, the assay can be adapted to measure 3'P (5'OH) DNA ends or breaks induced by oxidative DNA damaging agents by pretreatment of the DNA with alkaline phosphatase or Escherichia coli exonuclease III. The assay is capable of quantifying first several breaks per 10(5) bp.

Animals↗

PubMed1996

Apoptotic and anti-proliferative effects of fumonisin B1 in human keratinocytes, fibroblasts, esophageal epithelial cells and hepatoma cells.

Fumonisin B1 is associated with various animal and human carcinomas and toxicoses, including leukoencephalomalacia, hepatocarcinoma, pulmonary edema and esophageal carcinoma. We have examined the cellular effects of fumonisin B1 in vitro using cellular model systems relevant to potential human target tissues. Although fumonisin B1 has been described as a mitogen in Swiss 3T3 cells based on stimulation of [3H]thymidine incorporation, in the current work it was found that fumonisin B1 inhibited incorporation of [3H]thymidine by cultured neonatal human keratinocytes and HepG2 human hepatocarcinoma cells at 10(-7) and 10(-4) M respectively. Fumonisin B1 also inhibited clonal expansion of normal human keratinocytes and HET-1A human esophageal epithelial cells at 10(-5) M and growth in mass culture of normal human fibroblasts at 10(-7) M. The clonogenicity of normal human keratinocytes decreased to 45.5% of controls after exposure to 10(-4) M fumonisin B1 for 2 days. However, no differences in the cell cycle distribution of cultured keratinocytes was noted after exposure to 10(-5) M fumonisin B1 for 40 h. The viability of normal human keratinocytes and HET-1A cells decreased as a result of fumonisin B1 treatment, as determined by a fluorescein diacetate/propidium iodide flow cytometric cell viability assay. Fumonisin B1-treated keratinocytes released nucleosomal DNA fragments into the medium 2-3 days after exposure to 10(-4) M fumonisin B1 and increased DNA strand breaks were detected in attached keratinocytes exposed to 0-10(-4) M fumonisin B1 using a terminal deoxynucleotidyl transferase-based immunochemical assay system. Furthermore, fumonisin B1-treated keratinocytes and HET-1A cells developed morphological features consistent with apoptosis, as determined by phase contrast microscopy, fluorescent microscopy of acridine orange stained cells and electron microscopy. These results are consistent with the occurrence of fumonisin B1-mediated apoptosis in vitro.

3T3 Cells↗