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R Wiger

Publications and source records attributed to R Wiger.

At least 19 recordsLinked to original sources
PubMed2004

Differential distribution of splice variants of estrogen receptor beta in human testicular cells suggests specific functions in spermatogenesis.

A growing number of estrogen receptor beta (ER beta) splice variants are reported. Several of these have been discovered in testis, but with few exceptions little is known about their cellular localization. The aim of this study was to identify and elucidate the mRNA expression pattern of the different ER beta splice variants in human testicular cells. Northern analysis was performed on whole testis and fractions enriched in germ cells from untreated men and from estrogen-treated men undergoing sex change surgery. Probes were constructed in order to systematically screen for and identify various ER beta splice variants. Several ER beta bands were observed in the human testis, in which splice variants constituted the major part of total ER beta transcripts. Interestingly, only two ER beta wild-type transcripts were detected. These seem to be virtually absent from the haploid germ cells and are probably mainly located in somatic cells and/or primary spermatocytes. Several novel ER beta deletion variants were found in high levels in the haploid germ cell fractions and were nearly absent in testicular cells from the estrogen-treated men. The cell-dependent distribution raises the question whether splice variants may have specific functions in spermatogenesis, and whether the differential splicing of ER beta is regulated in a cell-specific manner.

Adolescent↗
PubMed2003

Mechanisms involved in the induction of apoptosis by T-2 and HT-2 toxins in HL-60 human promyelocytic leukemia cells.

T-2 and HT-2 toxins belong to a group of mycotoxins that are widely encountered as natural contaminants known to elicit toxic responses in hematopoietic cells. In the present study, HL-60 cells were used to characterize the apoptotic effects of T-2 and a major metabolite, HT-2, and to examine the mechanisms involved. Apoptotic cells were identified microscopically by chromatin condensation and nuclear fragmentation, by flow cytometric analysis, and by DNA gel electrophoresis. T-2 and HT-2 induced concentration-dependent apoptosis after 24 h in HL-60 cells, starting at concentrations of 3.1 and 6.25 ng/ml respectively. An increased number of apoptotic cells could be observed 4-6 h after exposure to 12.5 ng/ml of toxin. Little cytotoxicity (plasma membrane damage) was observed even after exposure to concentrations of toxins (25-50 ng/ml) inducing apoptosis in 60-100% of the cells. The apoptotic process was almost completely blocked in the presence of the general caspase inhibitor zVAD.fmk. In contrast, no or only minor effects were observed with the more specific caspase inhibitors DEVD.CHO, IETD.fmk, and DEVD.fmk. As judged by Western blotting, the levels of several procaspases (-3, -7, -8, -9, but not -12) were reduced 3-6 h after exposure to toxin. Substantial increases in the presumed active form(s) of caspase-8 and -9 were observed. Furthermore, poly(ADP-ribose) polymerase (PARP) was already markedly cleaved 3 h after toxin treatment, indicative of active caspase-3 and -7. No or only minor changes in Bcl-2, Bcl-XL and Bax levels were observed. BAPTA-AM and ZnCl2 blocked the degradation of procaspases, the fragmentation of PARP, and the induction of apoptosis. In summary, both T-2 and HT-2 induced apoptosis, with T-2 being somewhat more potent than HT-2. The divalent calcium concentration, [Ca2+], appears to be involved in the activation of several caspases, resulting in DNA fragmentation, chromosomal condensation, and nuclear fragmentation.

Apoptosis↗
PubMed2001

Highly efficient base excision repair (BER) in human and rat male germ cells.

The quality of germ cell DNA is critical for the fate of the offspring, yet there is limited knowledge of the DNA repair capabilities of such cells. One of the main DNA repair pathways is base excision repair (BER) which is initiated by DNA glycosylases that excise damaged bases, followed by incision of the generated abasic (AP) sites. We have studied human and rat methylpurine-DNA glycosylase (MPG), uracil-DNA glycosylase (UNG), and the major AP endonuclease (HAP1/APEX) in male germ cells. Enzymatic activities and western analyses indicate that these enzymes are present in human and rat male germ cells in amounts that are at least as high as in somatic cells. Minor differences were observed between different cellular stages of rat spermatogenesis and spermiogenesis. Repair of methylated DNA was also studied at the cellular level using the Comet assay. The repair was highly efficient in both human and rat male germ cells, in primary spermatocytes as well as round spermatids, compared to rat mononuclear blood cells or hepatocytes. This efficient BER removes frequently occurring DNA lesions that arise spontaneously or via environmental agents, thereby minimising the number of potential mutations transferred to the next generation.

Animals↗
PubMed2001

Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro.

The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, global genome repair (GGR) and transcription-coupled repair (TCR). In contrast to somatic cells, little is known regarding the expression of these pathways in germ cells. To address this basic question, we have studied NER in rat spermatogenic cells in crude cell suspension, in enriched cell stages and within seminiferous tubules after exposure to UV or N-acetoxy-2-acetylaminofluorene. Surprisingly, repair in spermatogenic cells was inefficient in the genome overall and in transcriptionally active genes indicating non-functional GGR and TCR. In contrast, extracts from early/mid pachytene cells displayed dual incision activity in vitro as high as extracts from somatic cells, demonstrating that the proteins involved in incision are present and functional in premeiotic cells. However, incision activities of extracts from diplotene cells and round spermatids were low, indicating a stage-dependent expression of incision activity. We hypothesize that sequestering of NER proteins by mispaired regions in DNA involved in synapsis and recombination may underlie the lack of NER activity in premeiotic cells.

Acetoxyacetylaminofluorene↗
PubMed2001

Seasonal changes in spermatogenic activity and in plasma levels of FSH, LH and testosterone, and the effect of immunization against inhibin in the male silver fox (Vulpes vulpes).

The cellular composition of the silver fox testis assessed by DNA flow cytometry and histological analysis exhibited marked circannual alterations. The proportion of haploid cells increased from late October to the breeding season in February, while that of diploid cells decreased and that of tetraploid cells fluctuated during the same period. Towards the end of March these changes were reversed. The seasonal variations in testicular histology paralleled the changes in distribution of cells from the different DNA populations. In August, 69% of the tubules contained spermatogonia as the only type of germ cell, while the remaining 31% also contained a few primary spermatocytes. In late October more than 50% of the tubules contained spermatocytes, and during the period of further activation from early December-February the seminiferous epithelium included round and/or elongated spermatids as well. In February, all tubules contained complete associations of germ cells, whereas in late March tubules with spermatogonia only and spermatogonia together with a few spermatocytes reappeared. In May, only such tubules could be found indicating total regression. Plasma concentrations of FSH and LH increased from early November, both gonadotrophins reaching maximum levels in December or early January, and then both declined during the second part of January, immediately prior to the actual breeding season. LH values showed a few smaller peaks in the beginning of June, whereas FSH levels were generally low until the next period of testicular reactivation. Testosterone concentrations were also low during most of the year but rose in November and December to reach a peak in January and a second peak in June. In animals immunized against inhibin the distribution of haploid, diploid and tetraploid cells did not deviate to any great extent from that in the controls, except in March when the immunized males had a markedly lower proportion of tetraploid cells, and in May, when they had a distinctly higher proportion of haploid cells. These findings were partly reflected by the histology. In the immunized animals, plasma FSH levels started to increase at approximately the same time but peaked higher and remained elevated almost 1 month longer than in the controls, whereas both the rise and decline in LH levels generally coincided with the variations in these animals, but the values were mostly higher. The testosterone profiles were similar to those in the controls except that the maximum values were also usually higher.

Animals↗
PubMed2001

Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis.

We investigated the frequency of spontaneous apoptosis and expression of the Bcl-2 family of proteins during normal spermatogenesis in man. Testicular tissue with both normal morphology and DNA content was obtained from necro-donors and fixed in Bouin's solution. A TdT-mediated dUTP end-labelling method (TUNEL) was used for the detection of apoptotic cells. Expression of apoptosis regulatory Bcl-2 family proteins and of p53 and p21(Waf1) was assessed by immunohistochemistry. Germ cell apoptosis was detected in all testes and was mainly seen in primary spermatocytes and spermatids and in a few spermatogonia. Bcl-2 and Bak were preferentially expressed in the compartments of spermatocytes and differentiating spermatids, while Bcl-x was preferentially expressed in spermatogonia. Bax showed a preferential expression in nuclei of round spermatids, whereas Bad was only seen in the acrosome region of various stages of spermatids. Mcl-1 staining was weak without a particular pattern, whereas expression of Bcl-w, p53 and p21(Waf1) proteins was not detected by immunohistochemistry. The results show that spontaneous apoptosis occurs in all male germ cell compartments in humans. Bcl-2 family proteins are distributed preferentially within distinct germ cell compartments suggesting a specific role for these proteins in the processes of differentiation and maturation during human spermatogenesis.

Adult↗
PubMed1998

Single-strand breaks, cell cycle arrest and apoptosis in HL-60 and LLCPK1 cells exposed to 1,2-dibromo-3-chloropropane.

We investigated 1,2-dibromo-3-chloropropane (DBCP)-induced DNA damage, cell cycle alterations and cell death in two cell lines, the human leukemia HL-60 and the pig kidney LLCPK1, both of which are derived from potential target sites for DBCP-induced toxicity. DBCP (30-300 micromol/L) caused a concentration-dependent increase in the levels of DNA single-strand breaks in both cell lines as well as in cultured human renal proximal tubular cells. After extended DBCP exposure in LLCPK1 cells (100 micromol/L, 30 h), the level of DNA breaks returned almost to control values. Incubation for 48 h showed a clear reduction of growth with DBCP concentrations as low as 10 micromol/L. Flow cytometric analysis showed that DBCP (1-10 micromol/L) exposure for 24 h caused an accumulation of LLCPK1 cells in the G2/M-phase. In HL-60 cells the accumulation in G2/M-phase was less marked, and at higher concentrations the cells accumulated in S-phase. Flow cytometric studies of HL-60 and LLCPK1 cells exposed to 100-500 micromol/L DBCP showed increased number of apoptotic cells/bodies with a lower DNA content than that of the G1 cells. Microscopic studies revealed that there were increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominant mode of death in these cell lines, following exposure to DBCP. The characteristic ladder pattern of apoptotic cells was observed when DNA from DBCP-treated HL-60 cells and LLCPK1 cells was electrophoresed in agarose. The finding that DBCP can cause an accumulation of cells in G2/M-phase and induce apoptosis in vitro may be of importance for the development of DBCP-induced toxicity in vivo.

Animals↗
PubMed1997

Apoptosis in HL-60 cells induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX).

The potent bacterial mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), which is formed during chlorination of drinking water, has been studied with respect to induction of cell death in promyelocytic leukemic HL-60 cells. Cells exposed to MX for 1 h and further incubated for 3 h, revealed no significant increase in the proportion of cells with compromised plasma membrane damage as judged by trypan blue or propidium iodide exclusion. However, flow cytometric studies and microscopic analysis of HL-60 cells after staining with Giemsa or Hoechst 33342, revealed that more than 30% of the cells exposed to 30-100 microM of MX, showed the characteristic morphology and biochemical markers of apoptosis. On the other hand, in cultures exposed to 300 microM MX, less than 5% of the cells appeared to be apoptotic (< G1 DNA) 3 h after treatment, which is similar to control values. Microscopic analysis of Hoechst 33342-stained cells revealed that they were 'arrested' in the early stages of chromatin condensation, but these cells eventually became necrotic. Some decrease in the percentage of cells in S-phase was observed 3 h after exposure to MX (10, 30 and 100 microM), but the induced cell death was not markedly cell stage specific. The characteristic ladder pattern of apoptotic cells was observed when DNA isolated from MX-exposed HL-60 cells was electrophoresed in agarose. The apoptotic process could also be detected by analysis with alkaline filter elution (AE), as a decrease in the total DNA recovered; and by single cell gel electrophoresis, as a decrease in the average number of cells/comets observable on each slide. With the protocols used no apparent increase in values in the normalized area above the curve (NAAC) (alkaline elution) or tail moments (single cell gel electrophoresis (SCGE)) were detected, indicating that apoptotic cells are not necessarily a confounding factor when assaying for genotoxicity with these techniques.

Adult↗
PubMed1997

Paracetamol inhibits cell cycling and induces apoptosis in HL-60 cells.

We investigated the effects of paracetamol on cell cycle and cell death in cultured HL-60 cells. Paracetamol (0.1-3.0 mM) caused a dose-dependent inhibition of cell proliferation. Inhibition of DNA synthesis was observed at as low concentrations as 0.03 mM. When HL-60 cells were exposed to paracetamol (0.1-1.0 mM), flow cytometric analysis showed that cells accumulated in the G1/S phase, and then slowly proceeded through the S-phase. Exposure to 2.0-3.0 mM paracetamol, on the other hand, resulted in a reduction of the number of cells in S-phase and G2/M phase, and a concurrent increase in cells/bodies with a lower DNA content than that of the G1 cells. Microscopic studies revealed increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominating mode of death in this cell line after exposure to paracetamol. However, when DNA from paracetamol-treated cells were electrophoresed in agarose, no clear ladder pattern characteristic for apoptotic cells was observed. This was most likely a result of secondary necrosis which followed incomplete apoptosis. Within 5-10 hr after start of paracetamol exposure, a marked downregulation of both c-myc and bcl-2 mRNA was observed. In conclusion, the present results show that exposure to high non-therapeutic concentrations of paracetamol cause cell cycle arrest and apoptosis in HL-60 cells in S-phase.

Acetaminophen↗
PubMed1996

The use of isolated lung cells in in vitro pulmonary toxicology: studies of DNA damage, apoptosis and alteration of gene expression.

Isolated lung cells constitute a valuable system for studying mechanisms involved in chemically induced toxicity in the lung. Different lung cells isolated from various species may be studied. Bronchiolar Clara and alveolar type 2 cells produce important lung-specific proteins, hold a major role in the metabolism of xenobiotics and serve as progenitor cells for other lung cell types. They are possible target cells in lung carcinogenesis. Alveolar macrophages play an important role in lung defence and in inflammatory responses. In the present study we have characterised chemically induced DNA damage, apoptosis, changes in cell cycle progression, transformation and alterations in gene expression in these specific lung cells isolated from rat, rabbit and human. Major differences between the cell types and the various species in the induction of DNA damage by chemicals were found, as measured by the 32P-postlabelling and alkaline filter elution techniques. Benzo(a)pyrene and hydrogen fluoride were found to induce apoptosis in the isolated cells as measured by microscopical analysis and flow cytometry. The function of various important tissue- or cell type specific proteins (CYP 2B1, Clara cell protein) and/or cellular signal transduction pathways constitute important targets that may be affected by exposure to toxic compounds. Using immunological and molecular techniques the differential expression of specific proteins/RNAs and their activity can be studied. Among other proteins, c/ebp is involved in the regulation of transcription at the end of signal pathways. The protein is differentially expressed in rat lung cells and thus could be suitable for studying differential toxic effects in various lung cells. In humans, bronchoalveolar lavage (BAL) fluid from human volunteers can be readily obtained and examined after exposure to different chemical compounds. An increase in the percentage of CD3-positive cells (T-lymphocytes) was found after exposure to hydrogen fluoride. The number of certain cell types and cytokines may be used to estimate the degree of inflammatory reaction. In conclusion, the use of in vitro data including the use of specific, primary human lung cell types may contribute considerably to the quality of risk assessment, together with in vivo data from animals and man.

Animals↗
PubMed1994

Effect of n-3 and n-6 fatty acids on proliferation and differentiation of promyelocytic leukemic HL-60 cells.

Promyelocytic leukemic HL-60 cells were incubated with different fatty acids. Arachidonic acid (AA; 20:4, n-6) and eicosapentaenoic acid (EPA; 20:5, n-3) were the most potent inhibitors of proliferation in a dose-dependent way. Retinoic acid (RA) was used as a positive control. Inhibitors of cyclooxygenase and lipoxygenase or addition of antioxidants did not influence the effect of EPA or AA on cell proliferation. Increased capacity to generate superoxide anions after phorbol ester treatment and a reduced serglycin messenger RNA level in cells treated with AA or EPA indicated that these fatty acids induced differentiation in HL-60 cells similar to that induced by RA. However, down-regulation of the c-myc mRNA level, also typical for differentiation with RA in HL-60 cells, was not observed in cells incubated with AA or EPA. Flow cytometric analyses showed that in cultures incubated with AA or EPA, the proportion of cells in the G1 phase of the cell cycle increased. Similar effects were observed with RA. By flow cytometry and light scatter analyses it could be shown that AA made 8% of the cells apoptotic and 7% necrotic. The corresponding numbers were 21% and 10% for RA-treated cells, and 19% and 32% for EPA-treated cells. The present study shows that AA and EPA reduce the proliferation rate of HL-60 cells. This is mediated by mechanisms independent of eicosanoids or lipid peroxidation products and is due to effects both on apoptosis/necrosis and cell differentiation.

Apoptosis↗
PubMed1990

Effects of ochratoxins A and B on prechondrogenic mesenchymal cells from chick embryo limb buds.

Prechondrogenic mesenchymal cells from the limb buds of 4-day chick embryos were cultured together with either ochratoxin A (OA) or ochratoxin B (OB) for 6 days. Both toxins inhibited the accumulation of cartilage proteoglycans and general protein synthesis in a concentration-related manner. The IC50 was 1.9 microM for OA and 6.2 microM for OB. Incubating the micromass with OA for periods ranging from 2 h up to 6 days did not produce any metabolites, indicating that OA was the proximal toxic compound in these studies. Neither preincubation with OB nor simultaneous exposure to non-toxic concentrations of OB together with various concentrations of OA influenced the toxicity of OA. The data indicate that interference of OA with general protein synthesis, both in vivo and in vitro, is an important mechanism underlying OA-induced embryotoxicity.

Animals↗
PubMed1989

Toxic effects of cyclophosphamide in differentiating chicken limb bud cell culture using rat liver 9,000 g supernatant or rat liver cells as an activation system: an in vitro short-term test for proteratogens.

Cells from 4-day chicken embryo limb buds plated as micromass cultures differentiate and form cartilage nodules after a 5- to 6-day growth period. The innate ability of these cells to biotransform compounds, such as cyclophosphamide (CP), into reactive metabolites is apparently inadequate. Protocols used rat liver S9 from Aroclor 1254-pretreated (PCB) rats or hepatocytes from control rats or polychlorinated biphenyls (PCB)-pretreated rats and were added to micromass cultures with CP causing concentration-related toxicity in the cell cultures. Coculturing chicken limb bud cells with PCB-hepatocytes was the most efficient method, yielding an IC50 of 2 micrograms CP per ml. Toxic CP metabolites accumulated in the medium of PCB-hepatocyte cultures and were quite stable. The toxicity of bioactivated CP was nearly identical for both proliferation and cartilage proteoglycan accumulation.

Animals↗
PubMed1988

Estimating chemical developmental hazard in a chicken embryo limb bud micromass system.

A chicken limb bud micromass system measuring the production of cartilage proteoglycans and the incorporation of radiolabelled thymidine and leucine was used to calculate potential developmental hazard. This hazard index was based upon the 50% inhibitory concentration (IC50) for these parameters and enabled classification of substances according to their intrinsic ability to inhibit differentiation at concentrations which were not cytotoxic. All-trans-retinoic acid, a potent teratogen, inhibited cartilage proteoglycan synthesis at a concentration which was more than 100 times lower than those affecting the incorporation of 14C-thymidine and 14C-leucine. On the other hand, sodium valproate, salicylate and isoniazid inhibited cartilage proteoglycan synthesis only near concentrations which were also cytotoxic. The results from testing with chicken limb bud micromass compared favorably, both with respect to effective concentrations and ratios describing developmental hazard to those from rodent micromass systems.

Animals↗
PubMed1985

Mosquito-borne arboviruses in Norway: further isolations and detection of antibodies to California encephalitis viruses in human, sheep and wildlife sera.

Seven virus strains antigenically related to the California encephalitis (CE) virus group were isolated from Norwegian Aedes spp. mosquitoes collected in 1976. So far CE viruses have been isolated from five different Aedes spp. in Norway. Furthermore, two virus strains related to the Bunyamwera group were isolated from Anopheles claviger. Antibodies to CE viruses were demonstrated in 22% of 1014 military recruits tested. Among 91 soldiers who were monitored by monthly blood samples during the mosquito season, seroconversions were detected in 11 individuals. Specific IgM antibodies were found in seven of them. Disease symptoms in connexion with the CE virus infections were not seen. The prevalence of CE antibodies in patients with CNS or respiratory infections was not higher than in control groups. Seroconversions were not seen in any of the groups. Screening of sheep sera from six different areas in northern Norway indicated significantly different degrees of CE virus activity. Passerine birds may be important CE virus hosts, while small rodents seem unimportant. Specific IgM antibodies were detected in the sera of one of three hares and one of two squirrels. Of the methods used, single radial haemolysis (SRH) and immuno-electro-osmophoresis (IEOP) seemed to be well suited for serological screenings. However, an indirect immunofluorescence antibody test (IFAT) which was used may be an attractive alternative if high-quality anti-species conjugates are available. The haemagglutination-inhibition (HI) test used gave a high number of false positive results.

Aedes↗