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H Marquardt

Publications and source records attributed to H Marquardt.

At least 19 recordsLinked to original sources
PubMed2002

Polyetheretherketone--cytotoxicity and mutagenicity in vitro.

The results of the incubation of polyetheretherketone (PEEK) fibre material with seven different genotype variants of salmonella bacterium showed with and without an external metabolic activation system (S9) with no mutagenic or cytotoxic activity of the test material. In the so-called "plate incorporation test" in which the PEEK raw material is finely cut and applied direct to the agar plate without the addition of solvent there was, as expected, no evidence of cytotoxic or mutagenic effects. In the HPRT test there was a significant increase in the number of mutants per dish, both after addition of N-acetylaminofluorene and N-methyl-N'-nitro-N-nitrosoguanidine (with and without an external metabolic activation system = +S9), but not after treatment of the cells with PEEK-DMSO-eluate. This means that the PEEK material under study did not release any substances that cause V79 cells to mutate. The investigation of the toxic reaction on the material under study revealed that the number of surviving colonies per 10(5) surviving cells lay within the range of or below the solvent control even in the presence of high PEEK concentrations (5.0 microg/ml). Therefore, in summary, the study produced no evidence of cell damage caused by PEEK.

2-Acetylaminofluorene↗
PubMed2002

Comparison of anthracycline-induced death of human leukemia cells: programmed cell death versus necrosis.

We investigated the mode of cell death induced by the anthracyclines, aclarubicin, doxorubicin and daunorubicin in the human leukemia cell lines, HL60 and Jurkat. The cells were incubated with drug concentrations up to 500 nM for periods between 3 and 24 hours, followed by morphological and biochemical analyses. All three substances induced DNA fragmentation, evident as DNA laddering and appearance of cells with hypodiploid DNA content, externalization of phosphatidyl serine, activation of caspases and degradation of the apoptosis-specific endonuclease inhibitor DFF45. However, concentrations and times necessary for these effects to occur were different, aclarubicin being the quickest acting drug with a lag phase of 3 h, followed by daunorubicin with 6 h and doxorubicin with 24 h. More importantly, aclarubicin induced these effects while the cell membrane was intact, whereas doxorubicin and daunorubicin led to immediate loss of membrane integrity. Programmed cell death is characterised by preservation of membrane integrity in order to allow removal of apoptotic bodies, whereas cell rupture is an early event in necrosis. We therefore suggest that, in our experimental settings, doxorubicin- and daunorubicin-induced cell death occurs by necrosis, while aclarubicin induces programmed cell death.

Aclarubicin↗
PubMed2001

Morphological transformation of C3H/M2 mouse fibroblasts by, and genotoxicity of, extracts of human milk.

Breast cancer may be initiated by environmental/dietary agents and human milk may act as an ex vivo indicator of in vivo exposure of mammary epithelial cells to genotoxins. Extracts of human milk from UK-resident women (n=7) were tested for their abilities to morphologically transform C3H/M2 mouse fibroblasts. Genotoxicities were assessed in the Salmonella typhimurium reverse-mutation assay in the presence of S9 using strains TA1538 and YG1019, and in metabolically-competent human MCL-5 cells with the micronucleus and with the alkaline single cell gel electrophoresis (comet) assays. Two of the seven extracts were inactive in the transformation assay both in the presence or absence of S9, two appeared to be equally transforming either in the presence or absence of S9, and two other extracts induced increased transformation frequencies in the presence of S9. A seventh extract, tested only in the absence of S9, was inactive. Extracts were either active or inactive in at least three of the four tests applied. Four extracts were active or inactive in all four tests. The results suggest that human milk could be used as a resource for investigations of the as-yet-unidentified transforming agents previously detected in mammary lipid.

Animals↗
PubMed2001

Cell transformation in vitro by food-derived heterocyclic amines Trp-P-1, Trp-P-2 and N(2)-OH-PhIP.

Heterocyclic aromatic amines (HCA) are formed upon frying of poultry, fish or meat and have been shown to induce tumours in rodent bioassays. We investigated the transforming activity of HCA in an in vitro assay using the M2/C3H mouse fibroblast cell line. An external metabolic activation system (rat liver homogenate) was required in order to observe any HCA-induced cytotoxic effects or cell transforming activity. Trp-P-1 and Trp-P-2 are shown to be among the most potent transforming HCA that have been detected in food. Metabolic activation of HCA has been shown to proceed via N-hydroxylation of the exocyclic amino group. Therefore, we tested 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N(2)-OH-PhIP) the activated metabolite of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. N(2)-OH-PhIP proved to be one of the most powerful compounds with transforming activity observable at a concentration as low as 30 nM. Since 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant HCA formed in fried and grilled food and N-hydroxylation appears to be the predominant pathway of human metabolism, these data support the hypothesis that HCA are involved in the aetiology of human cancer.

Amines↗
PubMed2000

Cell transformation and genotoxicity induced by bis(2, 3-dichloro-1-propyl) ether.

Bis(dichloropropyl) ether isomers have been identified in a petrochemical plant effluent through a toxicity identification evaluation study in the United States. They have also been observed in the microgram per liter range along one of the largest rivers in Europe, the Elbe River. In the present investigation, the genotoxic and transforming activity of a bis(dichloropropyl) ether isomer, bis(2,3-dichloro-1-propyl) ether, was assayed in vitro. The results demonstrate that bis(2,3-dichloro-1-propyl) ether is a potent mutagen in Salmonella typhimurium strains TA 100, TA 1535, and to a lesser extent in strain TA 98, but only when tested in the presence of a metabolic activation system (S9 mix). We have also investigated the induction of micronuclei by bis(2,3-dichloro-1-propyl) ether in the metabolically competent cell line, MCL-5. A linear, dose-dependent increase in micronuclei was observed following exposure to bis(2,3-dichloro-1-propyl) ether. The DNA strand-breaking capacity of this chemical was assessed in the alkaline single-cell gel electrophoresis ("comet") assay with MCL-5 cells. Bis(2,3-dichloro-1-propyl) ether clearly induced DNA strand breaks in the 4.5-45.5 microg/ml dose range. The ether also induced malignant transformation in C3H/M2 mouse fibroblasts after metabolic activation (S9 mix). Thus, it must be suspected that bis(2, 3-dichloro-1-propyl) ether may possess a carcinogenic potential. Since the compound along with its isomers is present in considerable concentrations in surface water, their elimination is a matter of significant public concern.

Animals↗
PubMed2000

Induction of cyclooxygenase expression and enhancement of malignant cell transformation by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The potential role of arachidonic acid metabolism in the enhancement (promotion) of malignant transformation of C3H/M2 mouse fibroblasts by the tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated using inhibitors of cyclooxygenase and lipoxygenase activities. The promoting effects of TCDD (1.5 pM) and of the reference tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA; 0.4 mM) on carcinogen (N-methyl-N'-nitro-N-nitrosoguanidine or 3-methylcholanthrene)-pre-treated fibroblasts was abolished by cotreatment with indomethacin, hydrocortisone, caffeic acid or nordihydroguaiaretic acid. A differential inhibition was found with N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide, a selective inhibitor of the cyclooxygenase isoenzyme COX-2: the promoting effect of TPA, but not that of TCDD, was abolished. Therefore, the role of the cyclooxygenase isoenzymes COX-1 and COX-2 during chronic exposure to TCDD was studied in more detail. Long-term treatment with TCDD (4-7 weeks) induced the expression of COX-1 and COX-2 mRNA in C3H/M2 fibroblasts (up to 2-fold). The enhanced expression of COX-2 protein in TCDD-treated fibroblasts was confirmed by western blot analysis. Concomitantly, the accumulation of the prostaglandins (PGs) PGE(2) and 6-keto-PGF(1alpha), which were identified as major metabolites of arachidonic acid in C3H/M2 cell cultures, was enhanced ( approximately 2-fold) following long-term treatment with TCDD (0.15 and 1.5 pM). The results suggest that the stimulation of arachidonic acid metabolism caused by a sustained cyclooxygenase induction is a critical event in the promoting action of TCDD in mouse fibroblasts in vitro. However, in contrast to TPA, the TCDD-mediated enhancement of malignant cell transformation may not specifically depend on the induction of COX-2 but, additionally, the induction of COX-1 activity may be necessary.

Animals↗
PubMed2000

Reduction in left ventricular messenger RNA for transforming growth factor beta(1) attenuates left ventricular fibrosis and improves survival without lowering blood pressure in the hypertensive TGR(mRen2)27 Rat.

Angiotensin II recruits transforming growth factor beta(1) (TGFbeta(1)) and is related to left ventricular fibrosis. However, it is unclear whether chronic in vivo reduction in left ventricular TGFbeta(1) expression blunts fibrosis and improves outcome in angiotensin II-dependent hypertension. Four-week-old male hypertensive TGR(mRen2)27 (Ren2) rats received either normal food, low-dose losartan (0.5 mg. kg(-1). d(-1)), or tranilast (a nonspecific TGFbeta inhibitor; 400 mg. kg(-1). d(-1)) (n=10 for each group) for 12 weeks and were compared with Sprague-Dawley control rats. The effect of tranilast on survival was evaluated in 34 additional untreated homozygous Ren2 rats. Tranilast or low-dose losartan did not lower blood pressure. However, the increase in left ventricular weight (Ren2 versus SD 3.1+/-0.16 versus 2.1+/- 0.06 mg/g body wt; P<0.05) was significantly (P<0.05) blunted by both tranilast (2.7+/-0.05) and losartan (2.7+/-0.07). Both drugs prevented the increase in left ventricular TGFbeta(1) mRNA and fibronectin mRNA and blunted the increase in hydroxyproline content and the increase in perivascular fibrosis. The perivascular fibrosis score correlated significantly with the level of expression of TGFbeta(1) (r=0.62; P=0.019). In situ hybridization demonstrated increases in TGFbeta(1) mRNA, predominantly in perivascular and nonmyocyte areas. Both drugs did not prevent the decrease in systolic or diastolic dP/dt, but tranilast significantly improved the survival of untreated Ren2 rats (P=0.029). In conclusion, TGFbeta(1) mRNA expression is increased predominantly in nonmyocyte regions in the hypertrophied left ventricle in this angiotensin II-dependent model of hypertension. This increase is probably due to high angiotensin II levels rather than to hypertension. This is the first study to suggest that chronic inhibition of TGFbeta(1) expression attenuates left ventricular hypertrophy and fibrosis, even without lowering blood pressure.

Animals↗
PubMed1999

Characterization of the heparan sulfate and chondroitin sulfate assembly sites in CD44.

Isoforms of CD44 are differentially modified by the glycosaminoglycans (GAGs) chondroitin sulfate (CS), heparan sulfate (HS), and keratan sulfate. GAG assembly occurs at serines followed by glycines (SG), but not all SG are utilized. Seven SG motifs are distributed in five CD44 exons, and in this paper we identify the HS and CS assembly sites that are utilized in CD44. Not all the CD44 SG sites are modified. The SGSG motif in CD44 exon V3 is the only HS assembly site; this site is also modified with CS. HS and CS attachment at that site was eliminated by mutation of the serines in the V3 motif to alanine (AGAG). Exon E5 is the only other CD44 exon that supports GAG assembly and is modified with CS. Using a number of recombinant CD44 protein fragments we show herein that the eight amino acids located downstream of the SGSG site in V3 are responsible for the specific addition of HS to this site. If the eight amino acids located downstream from the first SG site in CD44 exon E5 are exchanged with those located downstream of the SGSG site in exon V3, the SG site in E5 becomes modified with HS and CS. Likewise if the eight amino acids found downstream from the first SG in E5 are placed downstream from the SGSG in V3, this site is modified with CS but not HS. We also show that these sequences cannot direct the modification of CD44 with HS from a distance. Constructs containing CD44 exon V3 in which the SGSG motif was mutated to AGAG were not modified with HS even though they contained other SG motifs. Thus, a number of sequence and structural requirements that dictate GAG synthesis on CD44 have been identified.

Amino Acid Sequence↗
PubMed1999

Heterocyclic aromatic amines induce DNA strand breaks and cell transformation.

Heterocyclic aromatic amines (HAAs), formed during the cooking of foods, are known to induce tumours in rodent bioassays and may thus contribute to human cancer risk. We tested six HAAs in a morphological transformation assay and in three in vitro genotoxicity assays. The morphological transforming abilities of HAAs were tested, in the presence of rat-liver S9, in the C3H/M2 fibroblast cell line. Concentration levels of 50 microM 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 100 microM 2-amino-3,4,8-trimethylimidazo-[4,5-f]quinoxaline (4,8-DiMeIQx), 50 microM 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 100 microM 2-amino-9H-pyrido[2,3-b]indole (AalphaC), 100 microM 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and 15 microM 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced maximum transformation potencies of 5.5, 6.6, 6.3, 5.2, 7.3 and 9.2 transformed foci per 10(4) surviving cells, respectively. Bacterial mutagenic activity was determined in the presence of rat-liver S9 using the Salmonella typhimurium reverse-mutation assay employing strain YG1019. Mutagenic potencies of 3800 revertants (revs)/ng with 8-MeIQx, 2900 revs/ng with 4,8-DiMeIQx, 3480 revs/ng with IQ, 1.6 revs/ng with AalphaC, 2.9 revs/ng with MeAalphaC and 5 revs/ng with PhIP were observed. Clastogenic activity in vitro was analysed by the micronucleus assay in metabolically competent MCL-5 cells. Dose-dependent induction of micronuclei was observed for all HAAs tested with 1-5.4% of cells containing micronuclei at 10 ng/ml. Micronucleus induction was in the order 4,8-DiMeIQx > 8-MeIQx > IQ > MeAalphaC > PhIP > AalphaC. DNA strand-breaking activity in MCL-5 cells was measured by the alkaline single cell-gel (comet) assay. The lowest effect doses for significant increases (P < or = 0.0007, Mann-Whitney test) in comet tail length (microm) were 45.5 microg/ml (200 microM) for PhIP, 90.9 microg/ml (410-510 microM) for 4,8-DiMeIQx, IQ, MeAalphaC and AalphaC, and 454.5 microg/ml (2130 microM) for 8-MeIQx. It is not yet clear which of these assays most accurately reflects the genotoxic potential to humans of compounds of this class of environmental carcinogens.

Animals↗
PubMed1998

Identification of a binding site on Hsc70 for the immunosuppressant 15-deoxyspergualin.

Hsc70, the constitutive form of the heat shock protein 70 family of proteins, is involved in a number of biological activities which include protein folding and molecular chaperoning. Previously, we had shown that the immunosuppressant 15-deoxyspergualin (DSG) specifically interacted with Hsc70, as well as the Hsp90 family of proteins. Although the exact binding site on Hsc70 for protein substrates is unknown, a recent study shows that the extreme C-terminal four amino acids 647EEVD650 play a role in regulating AT-Pase activity, substrate binding, and interaction with HDJ-1. These four amino acids are also found at the C-terminus of Hsp90 and may be involved in similar functions. In this study, we show that DSG binds specifically to this EEVD regulatory domain. Binding of DSG to Hsc70 did not affect its ability to bind peptides. These results suggest that in addition to the ATP binding domain, there are two additional substrate binding domains on Hsc70. DSG should provide a tool for understanding the role of the EEVD motif in biological processes.

Adenosine Triphosphate↗
PubMed1998

Stimulation of the Ca2+-mediated egr-1 and c-fos expression in murine erythroleukaemia cells by cyclosporin A.

The Ca2+-induced expression of the primary response genes egr-1 and c-fos was investigated in the murine erythroleukaemia cell line ELM-I-1. Exposure of the cells to the Ca2+-ionophore A23187 led to a rapid transient rise in egr-1 and c-fos mRNA production followed by an increase in Egr-1 and c-Fos protein levels as well as an increase in Egr-1 and activator protein 1 (AP-1) DNA-binding activity. Preincubation of the cells with KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinases, strongly decreased the Ca2+-mediated expression of egr-1 and c-fos. In contrast, treatment with cyclosporin A, which inhibits the Ca2+/calmodulin-dependent protein phosphatase 2B or calcineurin, increased both egr-1 and c-fos mRNA production and the DNA-binding activity of the Egr-1 and AP-1 transcription factors in response to the intracellular Ca+ concentration ([Ca2+]i)-increasing agents A23187 or cyclopiazonic acid. Enhancement of the Ca2+-induced c-fos and egr-1 expression by cyclosporin A was correlated with the capability of this agent to inhibit calcineurin phosphatase activity in ELM-I-1 cells. Studies on the phosphorylation state and DNA-binding activity of the cAMP response element-binding protein (CREB) did not demonstrate an early Ca2+-dependent activation of this transcription factor, suggesting that the regulation of c-fos and egr-1 expression by Ca2+ is not linked to CREB in the haematopoietic ELM-I-1 cells. The results indicate that calcineurin exerts negative regulatory effects on both egr-1 and c-fos expression in murine erythroleukaemia cells, in addition to the calcineurin-mediated down-regulation of c-myb expression observed previously in this cell system. This study therefore emphasizes the important role of calcineurin as a negative modulator of gene expression in certain cell types.

Animals↗
PubMed1998

Morphological transformation of C3H/M2 mouse fibroblasts by extracts of human mammary lipid.

Mammary lipid may act as a reservoir for genotoxins. Mammary lipid extracts (MLEs), obtained from eight UK women (21-41 years) undergoing reduction mammoplasty, were examined for their abilities to morphologically transform C3H/M2 mouse fibroblasts. Resultant transformation rates were 0.27, 0.33, 0.07, 0.29, 0.21, 0.00, 0.07, and 0.13 transformed foci/treated dish, respectively. Although the lipid-extraction procedure used was originally designed to extract heterocyclic aromatic amines (HAAs), liquid chromatography/mass spectroscopy (LC/MS) with selective ion monitoring has failed to detect HAAs in any of the lipid extracts so far examined. Genotoxicities were also assessed in S. typhimurium TA98 and in metabolically competent human (MCL-5) cells by the micronucleus and by the alkaline single-cell gel ("comet") assays. The MLEs induced bacterial mutagenicity rates ranging from 0 to 498 revertants/plate/g-lipid equivalent and micronucleus-formation rates from 0 to 20 micronuclei/500 binucleate cells/g-lipid. Median comet tail lengths (induced with MLEs of 8.0 g-lipid equivalent) ranged from 6.0 to 74.0 micrometer. The results demonstrate the presence of as-yet-unidentified transforming agents in mammary lipid.

Adult↗
PubMed1998

HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences.

Previous studies showed that HLA-E was expressed in lymphoblastoid cell line (LCL) 721.221 cells, but surface expression was lacking. To determine the signals controlling surface expression, we constructed a series of hybrid genes using complementary portions derived from the HLA-E and HLA-A2 genes. In this manner, a hybrid of HLA-E was identified, designated AEH, which differed from HLA-E by having the HLA-A2 signal sequence substituting for the HLA-E leader peptide. Transfection of LCL 721.221 cells with AEH induced HLA-E surface expression. Analysis of peptides bound to HLA-E revealed that a nonamer peptide derived from the A2 signal sequence was the predominant peptide bound. LCL 721.221 cells transfected with certain class I genes, including HLA-G, were also sufficient to promote peptide binding and HLA-E surface expression without increasing the level of HLA-E heavy chain synthesis. Peptides bound to HLA-E consisted of nine amino acids, with methionine at position 2 and leucine in the carboxyl-terminal position, and were nearly identical to the leader sequence-derived peptide previously shown to be a predominant peptide bound to the murine Qa-1 Ag. Signal peptides derived from certain HLA-B proteins with threonine in position 2 only marginally up-regulated HLA-E surface expression in .221 cells. An examination of HLA-E peptide binding in the TAP negative cell line .134 indicated that peptide binding to HLA-E was dependent on a functional TAP heterodimer regardless of whether peptide was available in cis, as in the AEH construct, or in trans, as in the class I transfectants of .221 cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Cloning, overexpression, and purification of cytosine deaminase from Saccharomyces cerevisiae.

Cytosine deaminase is an enzyme which has been investigated for cancer chemotherapy as a result of its ability to convert the relatively nontoxic prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil. To facilitate investigations of the utility of cytosine deaminase for cancer chemotherapy, we have cloned and expressed the enzyme from Saccharomyces cerevisiae. The DNA sequence translates into a protein of 158 amino acids in length, with a predicted molecular weight of 17,563 kilodaltons. Alignment of the cytosine deaminase protein sequence from yeast with a variety of proteins defines a novel sequence motif of cytosine or cytidine binding enzymes. Recombinant expression cassettes encoding cytosine deaminase were transfected into monkey kidney COS cells, which lack endogenous cytosine deaminase, to test for production of a functional protein. Cell extracts from these transfectants contained detectable levels of enzyme activity capable of converting 5-fluorocytosine to 5-fluorouracil. Cytosine deaminase was expressed in yeast from a cDNA cassette under the control of an inducible promoter, increasing expression 250- to 300-fold relative to wild-type strains. A purification protocol has been developed which permits recovery of 60% of cytosine deaminase in active form from induced cell lysates after two purification steps. This protocol will be useful for isolating large quantities of pure enzyme which are required for the preclinical evaluation of monoclonal antibody-cytosine deaminase conjugates in combination with 5-fluorocytosine.

Amino Acid Sequence↗
PubMed1998

DNA adducts in human breast tissue: association with N-acetyltransferase-2 (NAT2) and NAT1 genotypes.

The etiology of human breast cancer is poorly understood, but circumstantial evidence points toward exogenous genotoxins as causative agents; they are believed to exert their carcinogenic action by binding to DNA. Because this binding is often preceded by metabolic activation, it is dependent on the expression and activities of metabolic enzymes of the host. Human mammary tissue samples from 42 women undergoing surgery for breast cancer or reduction mammoplasty were analyzed for DNA adducts by 32P-postlabeling analysis. With the butanol extraction method of DNA adduct enrichment, adduct levels were determined to be 0-414.6 adducts per 10(9) nucleotides, with considerable interindividual variation. To characterize the DNA adducts, we reanalyzed the adduct spots by reversed-phase high-performance liquid chromatography. Of two major adduct spots detected on TLC that accounted for up to 70% of the DNA modification, one eluted as a single peak on high-performance liquid chromatography, whereas the other was resolved into two distinct peaks of radioactivity. These major adducts were highly lipophilic in character. The N-acetyltransferase-1 (NAT1) and NAT2 genes were analyzed for common mutations using random RFLP analysis. An association between NAT2 acetylator status and adduct levels was observed; significantly elevated adduct levels occurred in the mammary DNA from women who were designated slow acetylators for NAT2 [median adduct level = 83.0 adducts per 10(9) nucleotides (range, 9.0-414.6)], as compared with the levels in individuals designated rapid acetylators for NAT2 [median adduct level = 39.7 adducts per 10(9) nucleotides (range, 0-91.0; P = 0.0053)]. On the other hand, NAT1 genotypes were not significantly associated with adduct levels. Although the agents responsible for the DNA modifications in the human breast are not known, this pilot study supports the hypothesis that DNA adduct formation in the human breast may be influenced by the NAT2 genotype.

Adolescent↗
PubMed1997

Pancreatic DNA adducts formed in vitro and in vivo by the food mutagens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alphaC).

Genotoxic heterocyclic amines have been detected in grilled or fried meat and tobacco smoke. Among these, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alphaC) have been shown to induce tumours in rodents in several organs. Here we report on the DNA adduct formation by PhIP and MeA alphaC in vitro and in vivo, both in rat hepatic and rat pancreatic tissues or cells. Using 32P-postlabelling analysis both compounds were shown to induce a dose-dependent DNA modification in primary rat hepatocytes that was correlated with cytotoxicity in these cells. In explanted rat pancreas maintained in dynamic short-term organ culture MeA alphaC was shown to induce covalent DNA adducts. No DNA adducts were observed with PhIP in this assay. DNA adducts were observed in the liver and the pancreas of F344 rats treated with PhIP, with a 36-times higher level of adducts in the pancreas, confirming data reported earlier. DNA adduct levels induced by feeding 32, 160 or 800 ppm MeA alphaC in the diet were dose-dependent and higher in the liver compared with other organs including pancreas. While for PhIP the N2-(desoxyguanin-8-yl)-derivative was accounting for more than 90% of DNA adducts detected, in the case of MeA alphaC the N2-(desoxyguanin-8-yl) adduct was predominant in vitro and determined in vivo as one of up to 5 DNA adducts. MeA alphaC had been reported to induce preneoplastic foci and tumours in the liver and tumours and atrophy in the pancreas. In the case of MeA alphaC, the DNA adduct formation and cytotoxicity observed by us in vitro and in vivo correlate with the organ specificity of the reported pathological lesions. In the case of PhIP our in vitro data in pancreas and liver and the low adduct levels in liver in vivo also reflect the reported lack of pathological effects in these organs. In contrast, in pancreas, in vivo extraordinarily high adduct levels induced by PhIP were observed confirming studies published earlier, in spite of the fact that this compound does not cause pancreatic lesions. This enigmatic observation is discussed and the relevant literature is reviewed.

Animals↗
PubMed1997

Signalling mechanisms in erythropoiesis: the enigmatic role of calcium.

The glycoprotein hormone, erythropoietin is the principal regulator of the production of circulating erythrocytes by controlling proliferation, differentiation and survival of its target erythroid progenitor cells. The receptor for erythropoietin is a type I cytokine receptor lacking intrinsic tyrosine kinase activity. It mediates tyrosine phosphorylation through its association with nonreceptor tyrosine kinases such as JAK2 and initiates a cascade of signalling events in response to erythropoietin. Significant progress has been made in identifying signalling pathways triggered by erythropoietin. However, the exact signalling mechanisms mediating the known physiological effects of erythropoietin in erythroid progenitor cells are poorly understood. There are many open questions including the role of Ca2+ in erythropoietin induced signal transduction. Although the results concerning the effect of erythropoietin on [Ca2+]i in various erythroid cells are conflicting, [Ca2+]i-increasing agents mimic the effect of erythropoietin on c-myb expression and activate the program of haemoglobin synthesis in murine erythroleukemia cells. An attempt is made in this review to survey recent data on the erythropoietin-induced signal transduction with respect to the different physiological effects of this hormone.

Animals↗