Paracetamol inhibits replicative DNA synthesis and induces sister chromatid exchange and chromosomal aberrations by inhibition of ribonucleotide reductase

Effects of paracetamol have been studied in a hydroxyurea (HU)-resistant mouse mammary tumour cell line TA3H2, shown to overproduce the small subunit of ribonucleotide reductase. These TA3H2 cells were much more resistant than the TA3H (wild-type) cells towards the inhibitory effect of paracetamol on cell growth, IC50 0.55 mM paracetamol for the wild-type compared to 2.7 mM for the HU-resistant cells. The reduced cell growth was due to an inhibition of replicative DNA synthesis, judged from an increased percentage of cells in S-phase measured by flow cytometry. Furthermore, in the wild-type cells, the increase in the number of cells in S phase was already observed at 0.1 mM while in the HU-resistant cell line this effect was first seen at 3.0 mM paracetamol. HU inhibits ribonucleotide reductase by destroying a tyrosyl free radical located on the small subunit of the enzyme. By electron paramagnetic resonance we demonstrate that paracetamol added to crude cell extracts of HU-resistant cells also immediately destroys this radical. These results show that paracetamol reduces DNA synthesis by a specific inhibition of ribonucleotide reductase. A concentration-dependent induction of sister chromatid exchanges was found both with paracetamol (1.0-10 mM) and HU (0.3-3 mM) in wild-type cells whereas no such increase was observed in HU-resistant cells. Paracetamol (1 mM for 2 h) also increased the number of chromosomal aberrations CAs in wild-type cells (i.e. chromatid breaks and chromatid exchanges). The frequency of CAs was not increased in HU-resistant cells at paracetamol concentrations up to 10 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the Environmental Oestrogens Bisphenol A,Tetrachlorobisphenol A,Tetrabromobisphenol A, 4‐Hydroxybiphenyl and 4,4′‐Dihydroxybiphenyl on Oestrogen Receptor Binding,Cell Proliferation and Regulation of Oestrogen Sensitive Proteins in the Human Breast Cancer Cell Line MCF‐7

Abstract: Bisphenol A is extensively used in the manufacturing of epoxy resins and polycarbonate plastics, whereas several brominated and chlorinated analogues are used as flame retardants and intermediates in the plastic industry. Due to the structural relationship between these chemicals and the high production volumes, we wanted to characterize and compare their potential oestrogen‐like potency using several end‐points in MCF‐7 cells: induction of pS2 protein and progesterone receptor, reduction of oestrogen receptor level, and stimulation of cell growth. Bisphenol A, tetrachloro‐ and tetrabromo‐bisphenol A, 4‐hydroxybiphenyl and 4,4′‐dihydroxybiphenyl all showed oestrogen‐like properties in MCF‐7 cells. The chemicals tested had affinity to the oestrogen receptor isolated from MCF‐7 cells, although their EC₅₀s were 1,000 to 80,000 times higher than the EC₅₀ of 17β‐oestradiol. Bisphenol A and 4‐hydroxybiphenyl induced cell growth in MCF‐7 cells, and the highest test concentrations induced responses, apparently exceeding the cell growth induced by 17β‐oestradiol. The other chemicals tested induced less than 50% of the maximum 17β‐oestradiol‐stimulated cell growth. Bisphenol A, 4‐hydroxybiphenyl, tetrabromobisphenol A and tetrachlorobisphenol A all increased the level of the oestrogen‐regulated proteins, progesterone receptor and pS2, whereas 4,4′‐dihydroxybiphenyl showed no such effect. Bisphenol A was the only chemical tested that clearly mimicked 17β‐oestradiol in its ability to reduce the level of cytosolic oestrogen receptors in MCF‐7 cells. By measuring several oestrogen‐dependent endpoints it seems that some xeno‐oestrogens cause an imbalanced oestrogen‐response. Their ability and potency in mimicking 17β‐oestrogen in one parameter is not necessarily accompanied by a similar effect in another oestrogen‐linked parameter.     

Effects of the environmental oestrogens bisphenol A,tetrachlorobisphenol A,tetrabromobisphenol A, 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl on oestrogen receptor binding,cell proliferation and regulation of oestrogen sensitive proteins in the human breast cancer cell line MCF-7

Bisphenol A is extensively used in the manufacturing of epoxy resins and polycarbonate plastics, whereas several brominated and chlorinated analogues are used as flame retardants and intermediates in the plastic industry. Due to the structural relationship between these chemicals and the high production volumes, we wanted to characterize and compare their potential oestrogen-like potency using several end-points in MCF-7 cells: induction of pS2 protein and progesterone receptor, reduction of oestrogen receptor level, and stimulation of cell growth. Bisphenol A, tetrachloro- and tetrabromo-bisphenol A, 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl all showed oestrogen-like properties in MCF-7 cells. The chemicals tested had affinity to the oestrogen receptor isolated from MCF-7 cells, although their EC50s were 1,000 to 80,000 times higher than the EC50 of 17beta-oestradiol. Bisphenol A and 4-hydroxybiphenyl induced cell growth in MCF-7 cells, and the highest test concentrations induced responses, apparently exceeding the cell growth induced by 17beta-oestradiol. The other chemicals tested induced less than 50% of the maximum 17beta-oestradiol-stimulated cell growth. Bisphenol A, 4-hydroxybiphenyl, tetrabromobisphenol A and tetrachlorobisphenol A all increased the level of the oestrogen-regulated proteins, progesterone receptor and pS2, whereas 4,4'-dihydroxybiphenyl showed no such effect. Bisphenol A was the only chemical tested that clearly mimicked 17beta-oestradiol in its ability to reduce the level of cytosolic oestrogen receptors in MCF-7 cells. By measuring several oestrogen-dependent endpoints it seems that some xeno-oestrogens cause an imbalanced oestrogen-response. Their ability and potency in mimicking 17beta-oestrogen in one parameter is not necessarily accompanied by a similar effect in another oestrogen-linked parameter.     

Effect of Sodium Fluoride on Protein and DNA Synthesis,Ornithine Decarboxylase Activity,and Polyamine Content in LS Cells

Abstract Sodium fluoride exhibited a dose dependent inhibitory effect on protein and DNA synthesis at concentrations from 1.3 mM in growing LS cells. The activity of ornithine decarboxylase (ODC) was slightly stimulated by 0.5 mM-NaF, but inhibited at 1.3 mM and above. The reduced enzyme activity seemed to be due to a reduced de novo formation of the enzyme caused by an inhibition of the protein synthesis. In spite of a reduction in ODC-activity, fluoride had no effect on the cellular polyamine content during the experimental period (10 hours).     

Toxic effects of paracetamol and related structures in V79 Chinese hamster cells

Exposure of V79 Chinese hamster cells to non-cytotoxic concentrationsof paracetamol (4-hydroxyacetanilide, 4-HAA) increased sisterchromatid exchange (SCE) in the absence of an external activationsystem. Furthermore, a selective inhibition of DNA synthesiswas observed at low 4-HAA concentrations. The inhibition couldbe counteracted by the addition of ascorbate, indicating thatthe effect is caused by an oxidation product of 4-HAA. In attemptto clarify possible relationships between cytotoxicity, inhibitionof DNA synthesis and increased SCE, we studied the effect of4-HAA and some related structures on these parameters. The relativeposistion of the amino group and the hydroxyl group on the aromaticringappear to be important for the inhibition of DNA synthesis.Removal of either of the two groups, N-acetylation and/or alkylationof the aromatic ring or phenolic oxygen decreased the effectof the aromatic amine on DNA synthesis. A significant responseon SCE was observed with 4-aminophenol, 4-HAA, 2-HAA, 3, 5-dimethyl-4-HAA,3-HAA and 2, 6-dimethyl-4-HAA (none of the other compounds weretested). The increase in SCE frequency caused by 4-HAA and itsanalogs does not seem to be related to more general cytotoxiceffects. The relative potencies of the compounds for SCE inductionparalleled, for the most part, their effects on DNA synthesis.However, the induction of SCE and the inhibition of DNA synthesisdid not occur at comparable concentrations. Thus, the possibilitythat 4-HAA increases the frequency of SCE through some othermechanism cannot be excluded.     

No effect of prolonged fluoride exposure on cytochrome P-450 and associated monooxygenases or on the level of polyamines in the rat

When exposing rats to drinking water containing 100 p.p.m. fluoride for 8 weeks, no effect could be detected in biochemical parameters of the liver, such as the concentrations of the polyamines putrescine, spermidine and spermine; the levels of microsomal protein and cytochrome P-450; or the activities of two associated monooxygenases, aryl hydrocarbon hydroxylase and ethylmorphine N-demethylase. Neither was there any increase in plasma glutamic-oxalacetic transaminase indicative of liver damage.     

Ornithine decarboxylase activity and polyamine content of normal and fluoride resistant LS cells.

The activity of ornithine decarboxylase (ODC) in suspension cultures of mouse fibroblasts, LS cells, varied in a characteristic cyclic pattern after dilution of the cultures. Two strains of fluoride resistant LS cells, FR6 and LSFR6 cells, exhibited the same cyclic pattern, but with markedly higher ODC activities. These fluoride resistant cells, however, contained less putrescine, the product of the ODC reaction. Possible reasons for this finding are discussed.     

Involvement of NADPH Oxidase and iNOS in Rodent Pulmonary Cytokine Responses to Urban Air and Mineral Particles

We have investigated the potential of two complex mineral particles (feldspar and mylonite), quartz (Min-U-Sil), and suspended particulate matter (SRM-1648) (SPM) from urban air to induce inflammatory cytokine responses in primary rat alveolar type 2 cells and alveolar macrophages, and the involvement of cellular formation of free radicals in these responses. All particle types induced an increased release of interleukin (IL)-6 and macrophage inflammatory protein (MIP)-2 from type 2 cells. Diphenyleneiodonium chloride (DPI), a selective inhibitor of NADPH-oxidase, reduced the IL-6 and MIP-2 responses to quartz, SPM and mylonite. N-(3-[Aminomethyl] benzyl) acetamidine (1400W), a selective inhibitor of inducible nitric oxide synthase (iNOS), significantly reduced the Il-6 response to SPM and feldspar in the type 2 cells. The macrophages displayed significantly increased TNF-α and MIP-2 release upon exposure to quartz or SPM. Here, DPI significantly reduced the tumor necrosis factor (TNF)-α and MIP-2 responses to quartz, and the MIP-2 response to SPM. No significant effect of 1400 W was detected in the alveolar macrophages. The role of particle-induced cellular generation of free radicals in lung cytokine responses was further elucidated in mice that lacked either NADPH-oxidase or iNOS as well as in wild-type (wt) mice. All particles were able to elicit increased cytokine levels in the bronchoalveolar lavage (BAL) fluid of the mice, although the levels depended on particle type. The NADPH-oxidase knockout (KO) mice demonstrated a significantly lower IL-6 and MIP-2 responses to SPM compared to their respective wt mice. The iNOS KO mice displayed significantly reduced IL-6, TNF-α, and MIP-2 responses to SPM. The overall results indicate the involvement of cellular free-radical formation in the pulmonary cytokine responses to particles of varying composition.     

Genotoxicity of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): formation of 2-hydroxamino-PhIP, a directly acting genotixic metabolite

Hepatocytes isdated from Aroclor 1254 (PCB) pretreated ratsmetabolized 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine(PhIP) to a reactive metabolite that induced DNA damage measuredby alkaline elution or as increased unscheduled DNA synthesis.PhIP induced mutations in Salmonella typhimuniun TA98 and DNAstrand breaks and sister chromatid exchange(s) in Chinese hamsterV79 cells co-incubated with PCR-hepatocytes. No, or only minorgenotoxic, effects were observed when hepatocytes from non-inducedrats were used. The bacterial mutagenicity could be inhibitedby -naphthoflavone, indicating a role of P-450 in the activationof PhIP. At least eight different metabolites could be separatedon HPLC after PhIP had been incubated with PCB-hepatocytes.All of the directly acting mutagenicity towards S.typhimuriumTA98 co-eluted with one of the metabolites. The identity ofthis metabolite was concluded to be 2-hydroamino-PhIP basedon the following evidence: (i) it reduced ferric ion to ferrousion as hydroxylamines do, (ii) it had an identical UV spectrumand chromatographic properties as a species formed upon redudionof 2-nitro-PhIP by NADPH P-450 reductase. This product displayeda major peak at m/z 241 during thermospray mass spectrometryin the positive-ion mode as would be expected from 2-hydroxamino-PhIP.2-Hydroxamino-PhIP was directly genotoxic both to TA98 and V79cells. The genotoxic activity of the medium after removing thehepatocytes remained stable for several hours. Compared to 2-amino-3,4-dimethylimidazo-[4,5-f]quinoline(MeIQ), PhIP caused a much larger increase in DNA damage inV79 cells (with hepatocyte activation), whereas MeIQ was morepotent with respect to DNA damage induced in hepatocytes andbacteria.