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.

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.

Environmental chemicals relevant for respiratory hypersensitivity: the indoor environment.

The allergenic constituents of non-industrial indoor environments are predominantly found in the biologic fraction. Several reports have related biological particles such as mites and their excreta, dander from pets and other furred animals, fungi and bacteria to allergic manifestations including respiratory hypersensitivity among the occupants of buildings. Also, bacterial cell-wall components and the spores of toxin-producing moulds may contribute to the induction of hypersensitivity, but the relevance for human health is not yet determined. The knowledge regarding hypersensitivity and asthmatic reactions after exposure to chemical agents is primarily based on data from occupational settings with much higher exposure levels than usually found in non-industrial indoor environments. However, there is evidence that indoor exposure to tobacco smoke, some volatile organic compounds (VOC) and various combustion products (either by using unvented stoves or from outdoor sources) can be related to asthmatic symptoms. In some susceptible individuals, the development of respiratory hypersensitivity or elicitation of asthmatic symptoms may also be related to the indiscriminate use of different household products followed by exposure to compounds such as diisocyanates, organic acid anhydrides, formaldehyde, styrene and hydroquinone. At present, the contribution of the indoor environment both to the development of respiratory hypersensitivity and for triggering asthmatic symptoms is far from elucidated.

International Commission for Protection against Environmental Mutagens and Carcinogens. An evaluation of the genetic toxicity of paracetamol.

During the last years, several reports have indicated genotoxic effects of paracetamol, a widely used non-prescription analgesic and antipyretic drug. Thus, a careful evaluation of a possible genotoxic effect related to paracetamol use is warranted. Studies in vitro and in vivo indicate that the reactive metabolite of paracetamol can bind irreversibly to DNA and cause DNA strand breaks. Paracetamol inhibits both replicative DNA synthesis and DNA repair synthesis in vitro and in experimental animals. Paracetamol does not cause gene mutations, either in bacteria or in mammalian cells. On the other hand, a co-mutagenic effect of paracetamol has been reported. Furthermore, paracetamol increases the frequency of chromosomal damage in mammalian cell lines, isolated human lymphocytes and experimental animals. Two independent studies have shown an increase in chromosomal damage in lymphocytes of human volunteers after intake of therapeutic doses of paracetamol, whereas a third study was negative. Paracetamol-induced chromosomal damage appears to be caused by an inhibition of ribonucleotide reductase. This indicates that a threshold level for the paracetamol-induced chromosomal damage may exist. Genotoxic effects of paracetamol have, however, been demonstrated both in vitro and in vivo at or near therapeutic concentrations. The data indicate that the use of paracetamol may contribute to an increase in the total burden of genotoxic damage in man. Thus, there may be a need to evaluate the therapeutic benefit of paracetamol, taking into consideration not only its potential to induce acute and chronic organ damage, but also genotoxic effects.

[DNA damages caused by paracetamol]. / DNA-skader av paracetamol.

Studies on cells in vitro and animal experiments show that paracetamol may bind covalently to DNA, inhibit DNA-replication and DNA-repair synthesis, and cause chromosomal aberrations in somatic cells. In two studies a higher level of chromosomal aberrations was found in lymphocytes after exposure of human volunteers to therapeutic doses of paracetamol, but in a third study the results were negative. Genotoxic effects of paracetamol have been demonstrated both in vitro and in vivo at or near therapeutic concentrations. Overall, the data indicate that paracetamol-use may contribute to an increase in the total burden of DNA-damage in man. Higher risk of cancer after exposure to paracetamol has not been demonstrated in epidemiological studies, nor has increased risk of cancer been unequivocally documented from animal experiments. It is generally accepted, however, that exposure that may cause DNA-damage in man should be reduced and if possible avoided. The therapeutic benefit of using paracetamol should therefore be carefully evaluated, taking into consideration its potential for inducing acute and chronic and genotoxic effects.

Comparative cytotoxic effects of acetaminophen (N-acetyl-p-aminophenol), a non-hepatotoxic regioisomer acetyl-m-aminophenol and their postulated reactive hydroquinone and quinone metabolites in monolayer cultures of mouse hepatocytes.

Toxic effects of acetaminophen (paracetamol, N-acetyl-p-aminophenol, APAP) in monolayer cultures of mouse hepatocytes developed over a period of 18 hr. N-Acetyl-m-aminophenol (AMAP) was approximately 10-fold less toxic than APAP, despite the fact that it bound covalently to a greater extent to hepatocyte macromolecules. AMAP did not deplete glutathione to as great an extent as APAP, indicating that their reactive metabolites may bind to different proteins or that oxidative damage in addition to arylation of proteins may be involved in the development of cell death. The toxicity of 3-methoxy-acetyl-p-aminophenol was similar to that of APAP, whereas the other hydroquinone and quinone metabolites were 8-10 times more cytotoxic than APAP. The potencies of these analogs were in the order: acetyl-m-aminophenol-p-benzoquinoneimine greater than or equal to 2,5-dihydroxyacetanilide greater than or equal to 3-methoxy-p-benzoquinone greater than or equal to N-acetyl-p-benzoquinone imine (NAPQI) greater than or equal to acetyl-m-aminophenol-o-benzoquinone greater than or equal to 3-hydroxy-acetyl-p-aminophenol. The relative toxic potencies of the hydroquinone and quinone metabolites of AMAP were comparable to that of NAPQI, and do not readily explain the marked difference between the cytotoxic effects of AMAP and APAP.

Genotoxic effects of the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in mammalian cells in vitro and in rats in vivo.

The potent bacterial mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]- furanone) (MX), which is formed during chlorination of drinking water and accounts for about one third of the Ames mutagenicity of tap water, has been studied with respect to its genotoxicity in vitro and in vivo. Treatment with 30-300 microM MX (1 h) induced DNA damage in a concentration-dependent manner in suspensions of rat hepatocytes, as measured by an automated alkaline elution system. The effect was similar in hepatocytes from PCB-induced and uninduced rats. DNA damage was induced in V79 Chinese hamster cells and in isolated rat testicular cells, at the same concentration level as in hepatocytes. Pretreating testicular cells with diethylmaleate, which depletes 85% of cellular glutathione, had no significant effect on the DNA damage induced by MX. The treatment conditions used in the alkaline elution experiments were not cytotoxic to any of the cell types used, as determined by trypan blue exclusion. V79 cells exposed to 2-5 microM MX (2 h) showed an increased frequency of sister-chromatid exchanges (SCE) whereas no significant effect on HGPRT mutation induction was observed. Higher concentrations (greater than 10 microM, 2 h) apparently blocked cell division. The data indicate that MX can react directly with DNA or that MX is metabolized to an ultimate mutagen via some enzyme which is common in mammalian cells. The in vivo experiments showed no evidence of genotoxicity after intraperitoneal (18 mg/kg, 1 h) or oral (18, 63 or 125 mg/kg, 1 h) administration of MX, as measured by alkaline elution, in any of the following organs: the pyloric part of the stomach, the duodenum, colon ascendens, liver, kidney, lung, bone marrow, urinary bladder and the testes. In conclusion, MX is a direct-acting genotoxicant in vitro but no in vivo genotoxicity was detected.

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)

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

Hepatocytes isolated from Aroclor 1254 (PCB) pretreated rats metabolized 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to a reactive metabolite that induced DNA damage measured by alkaline elution or as increased unscheduled DNA synthesis. PhIP induced mutations in Salmonella typhimurium TA98 and DNA strand breaks and sister chromatid exchange(s) in Chinese hamster V79 cells co-incubated with PCB-hepatocytes. No, or only minor genotoxic, effects were observed when hepatocytes from non-induced rats were used. The bacterial mutagenicity could be inhibited by alpha-naphthoflavone, indicating a role of P-450 in the activation of PhIP. At least eight different metabolites could be separated on HPLC after PhIP had been incubated with PCB-hepatocytes. All of the directly acting mutagenicity towards S.typhimurium TA98 co-eluted with one of the metabolites. The identity of this metabolite was concluded to be 2-hydroxamino-PhIP based on the following evidence: (i) it reduced ferric ion to ferrous ion as hydroxylamines do, (ii) it had an identical UV spectrum and chromatographic properties as a species formed upon reduction of 2-nitro-PhIP by NADPH P-450 reductase. This product displayed a major peak at m/z 241 during thermospray mass spectrometry in the positive-ion mode as would be expected from 2-hydroxamino-PhIP. 2-Hydroxamino-PhIP was directly genotoxic both to TA98 and V79 cells. The genotoxic activity of the medium after removing the hepatocytes remained stable for several hours. Compared to 2-amino-3,4-dimethylimidazo[4,5-f]quinolone (MeIQ), PhIP caused a much larger increase in DNA damage in V79 cells (with hepatocyte activation), whereas MeIQ was more potent with respect to DNA damage induced in hepatocytes and bacteria.

Short-term bioassays of fractionated emission samples from wood combustion.

Extracts of an emission sample from wood burning, consisting of particles and volatiles, have been fractionated on an HPLC silica gel column into five fractions of increasing polarity. Nonfractionated samples and the individual fractions have been tested in three different short-term bioassays: the Ames Salmonella assay, the sister chromatid exchange (SCE) induction-test in Chinese hamster ovary cells (CHO), and the cell transformation test on Syrian hamster embryo (SHE) cells. Most of the total activity was found in the volatile part of the sample with all three bioassays, whereas the particle extract had the highest activity per unit mass extracted. The second most polar fraction contained most of the mass and was also highly active in all assays. The most polar fraction was very potent in the Salmonella assay, but showed only a weak response in the eukaryotic bioassays. Storage of the samples for several months at 0 degrees C revealed that the bacterial mutagens present in the most polar fraction were labile; the mutagenicity was almost totally lost after 1 year's storage.

Cyclic AMP in urine, kidney and liver following long-term administration of fluoride to rats.

Rats were exposed to fluoride (F) for up to 8 weeks through the drinking water containing 100 p.p.m. F. Urine samples were regularly collected, together with tissue samples of liver and kidney at termination. All samples were assayed for cyclic AMP. No F-effect was found on liver or kidney levels of cyclic AMP. In the urine from the F-exposed rats there was an overall increase in the cyclic AMP concentration, but a decreased or unaffected 24 hrs urinary excretion of cyclic AMP. However, the F-exposed rats exhibited a considerably lower diuresis than the control animals. Because a significant negative correlation was found between diuresis and urinary concentration of cyclic AMP, the material was corrected to similarity in the mean diuresis between the two groups. Then no effect of F-exposure could be detected either in the urinary concentration or the daily excretion of cyclic AMP.

On the role of cyclic AMP in the cytotoxic effect of fluoride.

The possible role of adenosine 3',5'-monophosphate (cAMP) in the cytotoxic effect of fluoride was investigated in fluoride sensitive mouse fibroblasts (LS) and a subline of LS resistant to 6 mM fluoride (FR6). In both cell lines, growth was inhibited by dibutyryl-cAMP, prostaglandin (PG)E2 and theophylline, FR6 being somewhat more sensitive to these agents than LS. FR6 had lower basal cAMP levels in the intact cells and lower basal adenylate cyclase activity in the homogenate preparation than LS, but the percentual response of intact cells or adenylate cyclase preparations to PGE1 or PGE2 was about the same in the two cell lines, and the sensitivity of the adenylate cyclase to fluoride was similar. No measurable increase in cAMP content was found in either LS or FR6 after exposure of the intact cells to various concentrations of fluoride for various times. The present results indicate that the development of fluoride resistance in these cells is not due to decreased sensitivity to cAMP, and probably not due to altered cAMP-formation in response to fluoride. The growth inhibitory and cytotoxic effects of fluoride in LS cells is probably not mediated through cAMP.

Fluoride and energy metabolism in LS cells.

Six mM fluoride had no effect on lactate production in LS cells incubated in Eagle's minimum essential medium. In Krebs-Ringer phosphate buffer with 10 mM glucose inhibition was found with 3mM fluoride. The fluoride effect was similar in aerobic and anaerobic conditions. Twelve mM fluoride had no inhibitory effect on ATP levels in LS cells.