PubMed Health⌕ Search

Biomedical subjects

J A Holme

Publications and source records attributed to J A Holme.

At least 19 recordsLinked to original sources
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

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

Estrogen-like properties of brominated analogs of bisphenol A in the MCF-7 human breast cancer cell line.

Tetrabromobisphenol A (TeBBPA) is a four-meta-brominated variant of bisphenol A (BPA) and is one of the most commonly used brominated flame retardants worldwide. We compared the estrogenic potency of TeBBPA, BPA and the brominated analogs mono- (MBBPA), di- (DBBPA), and tribromobisphenol A (TrBBPA) in the estrogen-dependent human breast cancer cell line MCF-7. All of the compounds competed with 17beta-estradiol for binding to the estrogen receptor, although the affinity of the test chemicals to the estrogen receptor was much lower than that of 17beta-estradiol. TrBBPA and TeBBPA showed a considerably lower access to the estrogen receptors within intact MCF-7 cells incubated in 100% serum compared to incubation in serum-free medium, indicating a strong binding to serum proteins. BPA, MBBPA, and DBBPA showed only a slightly reduced access to the receptors. All of the test compounds induced proliferation in MCF-7 cells, the potential decreasing with increasing number of bromo-substitutions. TeBBPA did not induce maximal cell growth, indicating cytotoxic effects at high concentrations. BPA and the brominated analogs, except TeBBPA, induced progesterone receptor and pS2 to the same extent as 17beta-estradiol, although at much higher concentrations. Our studies demonstrate that compared to 17beta-estradiol, BPA and the brominated analogs have much lower estrogenic potencies for all of the endpoints tested, TeBBPA being the least estrogenic compound.

Benzhydryl Compounds↗
PubMed1999

[Possible carcinogenic risk associated with production and use of creosote-treated wood].

Creosote is a coal tar product which contains varying amounts of mutagenic and carcinogenic substances such as polycyclic aromatic hydrocarbons (PAHs) and benzene. Marketing and use of creosote and preparations containing creosote, as well as creosote-treated wood, are regulated by a EU Directive. According to the EU classification of such mixtures, inclusion of a warning against creosote as a carcinogen is not necessary if the contents of benzo[a]pyrene (BaP) and benzene are lower than 50 ppm (parts per million) and 1000 ppm, respectively. A recent well designed skin painting study in mice clearly indicates that the creosote preparations had a five-fold higher potency to induce skin cancer than the potency based on BaP content. Furthermore, it was estimated that creosote containing 50 ppm BaP would induce a significant incidence of skin cancer. Preliminary results from determination of concentrations of various carcinogens (BaP and benzene) in the air close to creosote impregnation plants as well as the crudely estimated exposure of children to dermal contact with creosote-treated wood, indicate that the life-time cancer risk from such exposures is in the order of one per 10,000. Despite the uncertainty related to such estimates, these risk levels give reasons for some concern. A further reduction in the content of PAH and benzene in creosote preparations should be considered.

Animals↗
PubMed1999

[Chlorination of drinking water--possible cancer risk from a by-product].

In a recent study, 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX; CAS no 77439-76-0) which is formed during chlorination of water containing organic substances, was found to be carcinogenic in the rat, at multiple sites in both sexes. MX is known as a potent bacterial mutagen. Epidemiological studies have suggested an association between chlorinated water consumption and a moderate increase in the risk of cancer. Although MX is a strong mutagen in prokaryotes, its genotoxic effects in mammalian cells are not so large, and more variable results are obtained. Very low concentrations of MX are found in drinking water (ng/L), whereas the genotoxic and carcinogenic effects in experimental animals of this compound are detectable at relatively high doses (mg/kg body weight). Relative to the risk for infectious diseases from the consumption of contaminated drinking water, the possible cancer risk associated with MX exposure appears to be low. Even so, efforts should be made to reduce disinfection byproduct formation by removing organic matter before chlorination.

Animals↗
PubMed1999

DNA damage induced by the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in mammalian cells in vitro and in mice.

3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) formed during chlorination of water containing natural organic substances, is a very potent bacterial mutagen. Recently, tumours at multiple sites were reported in rats given MX-containing drinking water. We have investigated the genotoxicity of MX in mammalian cells exposed in vitro and in vivo using alkaline filter elution to detect DNA single-strand breaks and/or alkali-labile sites (SSBs). Concentrations as high as 100 and 300 microM MX were required to induce detectable levels of SSBs in the HL-60 cells. If MX treatment was carried out in the presence of DNA repair inhibitors (AraC plus hydroxyurea), the sensitivity of the assay to detect MX-induced SSBs was increased by a factor of 100. The presence of serum proteins during exposure resulted in a minor reduction of the MX-induced DNA damage in HL-60 cells at the lowest MX concentrations. In primary cultures of testicular cells as well as in resting human peripheral blood mononuclear cells (PBMC), a slightly increased level of SSBs was observed at MX-concentrations above 30 microM, this effect was not further increased by repair inhibitors. In LLC-PK1 renal proximal tubular epithelial cells and in growth stimulated human peripheral PBMC, increased SSBs were detected at MX concentrations as low as low as 3-10 microM and higher using repair inhibitors, and at 10 times higher concentrations without repair inhibitors. No dose dependent DNA damage was detected in the liver, kidney, spleen and colon of male B6C3F1 mice administrated high doses of MX (40 and 80 mg kg-1). Moderately increased and dose dependent SSBs were detected in the liver and kidney in the presence of DNA repair inhibitors during MX treatment, but no such increase was observed in the spleen and colon.

Animals↗
PubMed1999

Characterization of metabolites of benz(j)aceanthrylene in faeces, urine and bile from rat.

1. The excretion of benz[j]aceanthrylene (B[j]A) and the biotransformation products found in faeces, urine and bile of rat exposed to [3H]-labelled B[j]A have been studied. 2. About 95% of the administered radioactivity was excreted within 7 days, 79% via faeces and 16% via urine, and most of the radioactivity in urine and faeces was excreted within 2 days. 3. The B[j]A metabolites excreted between days 1 and 2, including those excreted in bile during the first 5.5 h in a separate experiment, were further characterized by HPLC, UV and electrospray/atmospheric pressure chemical ionization mass spectrometry. 4. In faeces, bile and urine, hydroxylated B[j]A metabolites predominated. The major metabolites in faeces were B[j]A-1,2-dihydrodiol-8-hydroxy and B[j]A-1,2-dihydrodiol-10-hydroxy. These metabolites were found as conjugated metabolites in the bile. The glucuronide conjugate of B[j]A-1,2-dihydrodiol-10-hydroxy was also a major metabolite in urine. Two sulphate conjugates of oxidized B[j]A were detected in bile, a sulphate conjugate of a B[j]A-dihydrodiol-phenol and B[j]A-1,2-dihydrodiol-10-sulphate. Trans-B[j]A-1,2-dihydrodiol was detected in urine, faeces and bile. 5. These findings support the hypothesis that epoxidation at the cyclopenta ring is an important biotransformation pathway for B[j]A in vivo. In addition to the characterized metabolites, a large fraction of polar compounds, possibly glutathione conjugates, was also excreted in urine and bile.

Animals↗
PubMed1998

Metabolism and activation of cyclopenta polycyclic aromatic hydrocarbons in isolated human lymphocytes, HL-60 cells and exposed rats.

The metabolism of radiolabelled benz(j)aceanthrylene (B(j)A) was studied in suspensions of isolated human peripheral mononuclear blood cells (lymphocytes), using high performance liquid chromatography (HPLC). The only known metabolite found after 24 h exposure to 30 microg/ml (120 microM) B(j)A, was B(j)A-1,2-dihydrodiol, representing approximately 35% of the total metabolites formed. B(j)A, benz(l)aceanthrylene (B(l)A) and benzo(a)pyrene (B(a)P) all caused DNA adducts in human lymphocytes, as well as in the human promyelocytic cell line HL-60 cells, as measured by the 32P-postlabelling technique (30 microg/ml, 24 h). The total DNA adduct levels in human lymphocytes exposed to B(j)A, B(l)A or B(a)P were 0.13 +/- 0.03, 1.10 +/- 0.62 and 0.37 +/- 0.10 fmol/microg DNA, respectively, and similar levels were obtained in HL-60 cells (0.18 +/- 0.14, 0.97 +/- 0.35 and 0.29 +/- 0.17 fmol/microg DNA, respectively). For each compound, the human lymphocytes and HL-60 cells in addition showed similar DNA adduct patterns. Cells exposed to B(j)A revealed only one DNA adduct, which, judged by its TLC properties, resulted from B(j)A-1,2-epoxide. As measured by the alkaline filter elution technique, only low levels of single strand DNA breaks (SSB) were observed in both human lymphocytes and HL-60 cells after exposure to B(j)A, B(l)A or B(a)P (24 h, 30 microg/ml). By adding cytosine-1-beta-D-arabinofuranoside (Are C) and hydroxyurea (HU) 1 h prior to analysis to prevent strand break rejoining, some increase in SSB (2-3 times) was observed in the lymphocytes. Co-incubation of human lymphocytes with liver microsomes from PCB-treated rats for 1 h and exposure to B(j)A or B(l)A, increased the DNA adduct levels in the lymphocytes to 12.3 and 37.8 fmol/microg DNA, respectively. A large increase in SSB was also observed, whereas no such increase was observed after co-incubation with human liver microsomes. In vivo exposure of rats to 30 mg/kg B(j)A (i.p.) for 24 h revealed one major DNA adduct in lymphocytes and lung tissue (only one of three rats showed an adduct in liver tissue), apparently resulting from B(j)A-1,2-epoxide. The total DNA adduct level in the lymphocytes was 0.09 fmol/microg DNA, and in lung tissue between 0.10 and 0.62 fmol/microg DNA. Overall, the present data suggests that oxidation at the cyclopenta-ring is an important activation pathway for B(j)A in rats as well as in humans.

Animals↗
PubMed1998

Metabolism and activation of cyclopenta polycyclic aromatic hydrocarbons in liver tissue from rats and humans.

The metabolism of radiolabelled benz(j)aceanthrylene (B(j)A) was studied by high performance liquid chromatography (HPLC) using suspensions of hepatocytes and liver microsomes from control- or Aroclor 1254 (PCB)-treated rats, or with human liver microsomes (five different donors) as activation systems. The major metabolites formed in hepatocytes were sulfate conjugates, indicating that sulfation is an important detoxication pathway for B(j)A. In incubations with B(j)A and rat or human liver microsomes, the major metabolite formed was B(j)A-1,2-diol. Studies with rat liver microsomes using antibodies (Ab) towards either P4501A1, 1A2 or 3A2, resulted in approximately 30% reduction in covalent binding with all Ab-using microsomes from control animals, whereas with microsomes from PCB-treated animals an 85% reduction was observed using Ab towards P4501A2, and only minor reductions were observed with 1A1 or 3A2. When compared to B(j)A and benzo(a)pyrene (B(a)P), benz(1)aceanthrylene (B(l)A) caused higher numbers of revertants in the Salmonella assay when plated with rat liver microsomes from control animals or human liver microsomes. The total DNA adduct levels in hepatocytes from control animals after 2 h exposure to 30 micrograms/ml (120 microM) B(j)A or B(l)A, as measured by the 32P-postlabelling technique, were 3.8 +/- 1.5 and 10.1 +/- 5.8 fmol/microgram DNA, respectively. PCB-treatment decreased the total level of B(j)A adducts slightly (1.8 +/- 0.5 fmol/microgram DNA), whereas in contrast the level of B(1)A adducts was increased (24.5 +/- 20.1 fmol/microgram DNA). The major DNA adduct formed in control hepatocytes exposed to B(j)A co-chromatographed with B(j)A-1,2-oxide, which also appeared to be the major adduct formed when rat or human liver microsomes were co-incubated with calf thymus DNA. The total DNA adduct levels in the modified calf thymus DNA after 30 min exposure to 30 micrograms/ml B(j)A, B(l)A or B(a)P using rat liver microsomes form control animals, were 3.6, 66.3 and 1.4 fmol/microgram DNA, respectively. These levels increased to 22.7, 93.3 and 7.4 fmol/microgram DNA, respectively, using microsomes from PCB-treated animals. With human liver microsomes, the total DNA adduct levels after exposure to B(j)A, B(l)A or B(a)P, ranged between 0.4-1.0, 0.3-4.3, and 0.1-0.3 fmol/microgram DNA, respectively. Overall, the present data supports the notion that oxidation at the cyclopenta-ring is an important activation pathway for B(j)A, and indicate that the activation mechanism for B(j)A is similar in rat and human liver tissue.

Adolescent↗
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

DNA damage, gadd153 expression, and cytotoxicity in plateau-phase renal proximal tubular epithelial cells treated with a quinol thioether.

2-Bromo-bis-(glutathion-S-yl)hydroquinone [2-Br-bis-(GSyl)HQ] causes DNA single-strand breaks (SSB), causes growth arrest, induces the expression of gadd153 (a gene inducible by growth arrest and DNA damage), and decreases histone H2B mRNA in log-phase renal proximal tubular epithelial cells (LLC-PK1). Renal epithelial cells in vivo normally exhibit a low mitotic index, therefore experiments in both plateau- and log-phase cells are necessary for a comprehensive understanding of the stress response to 2-Br-bis-(GSyl)HQ. In the present article we demonstrate that not all features of the stress response in log-phase cells are reproduced in plateau-phase cells. Thus, although 2-Br-bis-(GSyl)HQ causes concentration and time-dependent increases in DNA SSB, and increases the expression of gadd153, histone H2B mRNA levels are unaltered in plateau-phase cells. The relationship between reactive oxygen species, DNA damage, gene expression, and cytotoxicity was also investigated. Our findings suggest that (i) 2-Br-bis-(GSyl)HQ-mediated DNA damage in LLC-PK1 cells is mediated by the generation of H2O2; (ii) DNA damage, either directly or indirectly, contributes to cell death; and (iii) DNA damage, either directly or indirectly, provides the initial signal for gadd153 expression. In addition, DNA repair is rapid in LLC-PK1 cells, and the DNA-repair inhibitors 1-beta-D-arabinofuranosylcytosine and hydroxyurea have no effect on the amount of DNA SSB. Although the addition of 3-aminobenzamide following 2-Br-bis-(GSyl)HQ exposure has no effect on the removal of DNA SSB, it causes a slight but significant increase in gadd153 expression and cell viability, indicating that activation of poly(ADP-ribose)polymerase may exacerbate toxicity. Finally, aurintricarboxylic acid did not prevent DNA SSB or cytotoxicity in 2-Br-bis-(GSyl) HQ-treated LLC-PK1 cells, implying that activation of endonucleases does not play a role in these processes.

Animals↗
PubMed1997

DNA damage induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in HL-60 cells and purified DNA in vitro.

Chlorinated tap water often contains 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), which is a potent directly acting bacterial mutagen. We have investigated the induction of DNA damage by MX in a promyelocytic human leukaemia cell line (HL-60 cells). Exposure of HL-60 cells to 100-300 microM MX resulted in increased levels of DNA single-strand breaks and/or alkali-labile sites (SSBs) as detected by alkaline filter elution. When adding inhibitors of DNA break repair (AraC plus hydroxyurea), increased levels of DNA SSBs were observed at very low concentrations (1-3 microM) of MX, as observed by both alkaline filter elution and the single-cell gel electrophoresis assay. Increased DNA SSBs could also be observed if DNA repair inhibitors were added immediately after exposure to 10 microM MX, indicating that low concentrations of MX cause a relatively stable modification of DNA that may be recognized and incised by DNA repair enzyme activities. Further studies with DNA break repair inhibitors indicated that HL-60 cells exposed to 10 microM MX for 1 h repaired 50% of their initial DNA damage during a 2-h period and the repair appeared to be complete at 22 h. Analysis of MX-treated DNA by sequencing methods indicated that MX preferentially reacts with guanines in DNA.

Cytarabine↗
PubMed1997

[Are environmental chemicals with hormone-like properties a health problem?].

Lately, a hypothesis linking higher frequency of testicular cancer, reduced semen quality and malformation of the male sexual organs with increased embryonic/foetal exposure to oestrogenic chemicals has received wide attention in the media. There are several examples where point-source chemical pollution has been convincingly associated with endocrine-related changes in wildlife. Such changes have also been reproduced in experimental studies. There is very little evidence, however, of such an association in humans. Nevertheless, the findings provide a clear challenge to toxicologists and epidemiologists in order to elucidate possible public health risks from environmental exposure to chemicals with hormone-like effects. Better test strategies and reproductive toxicity test guidelines are needed in order to assess any such risks, and to provide a basis for possible regulatory action.

Animals↗
PubMed1997

Genotoxic effects of cyclopenta-fused polycyclic aromatic hydrocarbons in different types of isolated rat lung cells.

The genotoxic effects of the environmental contaminants benz[j]aceanthrylene (B[j]A), benz[l]aceanthrylene (B[l]A) and benzo[a]pyrene (B[a]P), and the metabolism of radiolabelled B[j]A, were studied using rat lung microsomes and various types of isolated rat lung cells from control and Aroclor 1254 (PCB) treated animals. All three compounds (10 or 20 microg/plate) resulted in low, but detectable, levels of His+ revertants in the Salmonella assay when plated with control lung microsomes. The two cyclopenta polycyclic aromatic hydrocarbons (CP-PAH) B[j]A and B[l]A, gave increased levels of revertants when plated with microsomes from PCB-treated animals. Clara cells, type 2 cells and alveolar macrophages isolated from control rats were exposed to B[j]A, B[l]A or B[a]P (30 microg/ml, 1 h), but neither of the cell types showed any DNA damage when measured by alkaline filter elution. However, both B[j]A and B[l]A (30 microg/ml, 2 h) caused DNA adducts in all three cell types, measured by the 32P-post-labelling technique, whereas no B[a]P adducts were detected (30 microg/ml, 2 h). The total DNA adduct levels in Clara cells, type 2 cells and macrophages exposed to B[j]A were 0.085 +/- 0.033, 0.053 +/- 0.001 and 0.170 +/- 0.030 fmol/microg DNA, respectively, whereas the total levels in cells exposed to B[l]A were 0.140 +/- 0.070, 0.140 +/- 0.030 and 0.220 +/- 0.080 fmol/microg DNA, respectively. Cells exposed to B[j]A revealed only one adduct which corresponds with the B[j]A-1,2-oxide DNA adduct. Judged from high performance liquid chromatography (HPLC) analysis using radiolabelled B[j]A (30 microg/ml, 30 min), the major metabolite formed in control microsomes was B[j]A-1,2-diol. Thus, oxidation at the cyclopenta ring appears to be the most important activation pathway for B[j]A with control rat lung cells. Exposure of lung cells to CP-PAH (30 microg/ml, 2 h) isolated from PCB pretreated rats resulted in slightly increased DNA adduct levels in Clara cells and macrophages when compared to cells isolated from control rats. Furthermore, the adduct pattern had shifted, and no apparent B[j]A-1,2-oxide adduct could be detected on the thin layer chromatography (TLC) plate. In contrast, the major metabolite formed with microsomes from PCB-treated animals was still the B[j]A-1,2-diol.

Animals↗
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

Effects of phenethyl isothiocyanate on metabolism and on genotoxicity of dimethylnitrosamine and 2-amino-1-methyl-6-phenylimidazo[4, 5-beta]pyridine (PhIP).

Phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables, inhibited the genotoxic effects of N-nitrosodimethylamine (DMN) and of 2-amino-1-methyl-6-phenylimidazo[4,5-beta]pyridine (PhIP) in differential DNA repair assays with E. coli K-12 strains in vitro and in animal mediated assays with mice. In Salmonella typhimurium, the mutagenic activities of DMN and PhIP measured after activation with S-9 homogenates from several organs of PEITC-treated mice were substantially lower than those obtained with homogenates of untreated animals as well. PEITC also reduced the formation of micronuclei by DMN in metabolically competent Hep-G-2 cells of human origin but was ineffective in combination with PhIP. Biochemical investigations showed that the prevention of genotoxic effects of DMN by PEITC results form an inhibition of its alpha-hydroxylation. The effect of oral administration of PEITC on the formation of DNA adducts of PhIP was examined in the colon and liver of mice. No inhibition of adduct formation was observed in these experiments. Biochemical experiments showed that PEITC reduces not only the metabolic activation of PhIP via 2-hydroxyamino PhIP but also inhibits a detoxification pathway (formation of 4-hydroxy PhIP). The present results can be taken as an indication that the anticarcinogenic activities of isothiocyanates towards nitrosamines are paralleled by antimutagenic effects, and that probably no such protective effects occur in combination with heterocyclic amines. Furthermore, our findings show that the effects of chemopreventive agents demonstrated in bacteria in vitro cannot always be extrapolated to reactions occurring in intact mammalian cells.

Animals↗