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Several earlier studies have shown the presence of more dust and allergens in carpets compared with non-carpeted floors. At the same time, adverse effects of carpeted floors on perceived indoor air quality as well as worsening of symptoms in individuals with asthma and allergies were reported. Avoiding extensive carpet use in offices, schools, kindergartens and bedrooms has therefore been recommended by several health authorities. More recently, carpet producers have argued that former assessments were obsolete and that modern rugs are unproblematic, even for those with asthma and allergies. To investigate whether the recommendation to be cautious with the use of carpets is still valid, or whether there are new data supporting that carpet flooring do not present a problem for indoor air quality and health, we have reviewed the literature on this matter. We have not found updated peer reviewed evidence that carpeted floor is unproblematic for the indoor environment. On the contrary, also more recent data support that carpets may act as a repository for pollutants which may become resuspended upon activity in the carpeted area. Also, the use of carpets is still linked to perception of reduced indoor air quality as well as adverse health effects as previously reported. To our knowledge, there are no publications that report on deposition of pollutants and adverse health outcomes associated with modern rugs. However, due to the three-dimensional structure of carpets, any carpet will to some extent act like a sink. Thus, continued caution should still be exercised when considering the use of wall-to-wall carpeted floors in schools, kindergartens and offices, as well as in children's bedrooms unless special needs indicate that carpets are preferable., Competing Interests: The authors declare no conflict of interest.

We have characterized cell death in THP-1 cells after exposure to heat-treated spores from satratoxin G-producing Stachybotrys chartarum isolate IBT 9631, atranone-producing S. chartarum isolate IBT 9634, and sterigmatocystin-producing Aspergillus versicolor isolate IBT 3781, as well as the trichothecenes T-2 and satratoxin G. Spores induced cell death within 3-6 h, with Stachybotrys appearing most potent. IBT 9631 induced both apoptosis and necrosis, while IBT 9634 and IBT 3781 induced mostly necrosis. T-2 toxin and satratoxin G caused mainly apoptosis. Comet assay +/- formamidopyrimidine DNA glycosylase showed that only the spore exposures induced early (3h) oxidative DNA damage. Likewise, only the spores increased the formation of reactive oxygen species (ROS), suggesting that spores as particles may induce ROS formation and oxidative DNA damage. Increased Ataxia Telangiectasia Mutated (ATM) phosphorylation, indicating DNA damage, was observed after all exposures. The DNA damage response induced by IBT 9631 as well as satratoxin G was characterized by rapid (15 min) activation of p38 and H2AX. The p38 inhibitor SB 202190 reduced IBT 9631-induced H2AX activation. Both IBT 9631 and T-2 induced activation of Chk2 and H2AX after 3 h. The ATM inhibitor KU 55933, as well as transfection of cells with ATM siRNA, reduced this activation, suggesting a partial role for ATM as upstream activator for Chk2 and H2AX. In conclusion, activation of Chk2 and H2AX correlated with spore- and toxin-induced apoptosis. For IBT 9631 and satratoxin G, additional factors may be involved in triggering apoptosis, most notably p38 activation.

Phthalates are found in numerous consumer products, including interior materials like polyvinyl chloride (PVC). Several studies have identified phthalates in indoor air. A recent case-control study demonstrated associations between allergic symptoms in children and the concentration of phthalates in dust collected from their homes. Here we have analyzed the content of selected phthalates in particulate matter (PM): PM(10) and PM(2.5) filter samples collected in 14 different indoor environments. The results showed the presence of the phthalates di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP), dicyclohexyl phthalate (DCHP) and diethyl hexyl phthalate (DEHP) in the samples. The dominating phthalate in both PM(10) and PM(2.5) samples from all locations was DBP. More than a 10-fold variation in the mean concentration of total phthalates between sampling sites was observed. The highest levels of total phthalates were detected in one children's room, one kindergarten, in two primary schools, and in a computer room. The relative contribution of total phthalates in PM(10) and PM(2.5) was 1.1 +/- 0.3% for both size fractions. The contribution of total phthalates in PM(2.5) to total phthalates in PM(10) ranged from 23-81%, suggesting different sources. Of the phthalates that were analyzed in the PM material, DBP was found to be the major phthalate in rubber from car tyres. However, our analyses indicate that tyre wear was of minor importance for indoor levels of both DBP as well as total phthalates. Overall, these results support the notion that inhalation of indoor PM contributes to the total phthalate exposure.

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-alpha and MIP-2 release upon exposure to quartz or SPM. Here, DPI significantly reduced the tumor necrosis factor (TNF)-alpha 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-alpha, 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.

The development of various in vitro screening methods has led to identification of novel estrogenic chemicals of natural and anthropogenic origin. In this study, the (anti)estrogenic potential of several environmental chemicals were compared in an array of in vitro test systems comprising: (i) competitive binding to estrogen receptors derived from the human breast cancer cell line MCF-7 (hER) and rainbow trout (Oncorhynchus mykiss) (rtER), (ii) a proliferation assay with MCF-7 cells (E-SCREEN), and iii) induction of vitellogenin (rtVtg) in isolated rainbow trout hepatocytes. The results showed substantial differences in assay sensitivity for potent estrogens like 17beta-estradiol, diethylstilbestrol and zearalenone (ranking order of sensitivity: E-SCREEN > hER approximately rtER approximately rtVtg). Chemicals like 4-n-nonylphenol and bisphenol A had higher relative binding affinity to the hER, whereas 4-t-butylphenol and 4-n-butylphenol showed highest affinity to the rtER. Zearalenone and the novel estrogen 4-t-butylhexanol displayed a considerable higher relative potency in the E-SCREEN than the rtVtg assay, whereas alkylphenols and the novel estrogen mimic 4-t-butyl-nitrobenzene were most potent in fish cells. Correlation analysis of data from the test systems suggest that interspecies differences is largely due to inter-assay variation of the ER-dependent cellular responses, whereas binding to the ER are fairly similar in the two species tested.

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.

A large number of halogenated phenols are detected in the blood of humans, fish and wild-animals. We have characterized the estrogen-like activity of phenol, 4-bromophenol (4-BP), 2,4-dibromophenol (2,4-DBP), 2,4,6-tribromophenol (2,4,6-TBP) and 4-tert-butylphenol (tert-BP) using the estrogen-dependent human breast cancer cell line MCF-7. 4-BP, 2,4-DBP and 4-tert-BP all bind to the estrogen receptor (ER) with approximately 10,000-fold less affinity than 17 beta-estradiol (17 beta-E). 2,4,6-TBP was only able to displace 43% of radiolabelled estrogen when tested at concentrations up to 1 microM, whereas phenol had no affinity for the ER. 4-tert-BP stimulated cell growth and induced estrogen-regulated proteins such as the progesterone receptor (PgR) and pS2. The brominated phenols, however, although binding to the ER, did not stimulate cell growth or increase the levels of the PgR or pS2, or reduce the level of 17 beta-E induced pS2. On the contrary, 4-BP, 2,4-DBP and partly 4-tert-BP reduced 17 beta-E-stimulated cell growth apparently by an ER independent mechanism.

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.

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.

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.

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.

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.

Paracetamol blocks DNA replication by inhibiting deoxyribonucleotide (dNTP) synthesis and may therefore also interfere with DNA repair. In the present work various mammalian cell types were treated with genotoxic agents and allowed to repair in the presence or absence of paracetamol. Alkaline elution was used to assay DNA single-strand breaks plus alkali-labile sites (= SSBs). Resting human mononuclear blood cells (MNC) exposed to 4-nitroquinoline N-oxide (NQO, 3 microM) plus 0.3 mM paracetamol contained twice as many DNA SSBs compared to MNC exposed to NQO alone, and the level of SSBs decreased more slowly during repair in the presence of paracetamol. Deoxyribonucleosides reversed the effects of paracetamol. SSBs induced by MMS or X-rays (2.6 Gy) were not increased by paracetamol. Resting and growth-stimulated MNC, HL-60 cells, rat hepatocytes and human fibroblasts exposed to UV-C (3-12 J/m2) showed varying levels of transient SSBs formed during repair but these were consistently higher in the presence of paracetamol (0.3-1 mM). In rat testicular cells SSBs were induced by NQO and the levels were further increased in the presence of paracetamol, whereas after UV almost no SSBs were detected during repair. The cell-type specific levels of transient SSBs after UV did not correlate with the rate of incision of DNA lesions, measured as the rate of SSB accumulation in the presence of repair inhibitors Ara C plus hydroxyurea. Transient SSBs were present in resting MNC for at least 24 h after UV and paracetamol increased these breaks 4-fold however the overall rate of removal of excisable photodamage during repair did not appear to be reduced by the presence of paracetamol. The present data indicate that paracetamol interferes with nucleotide excision repair in several mammalian cell types. This constitutes a mechanism by which paracetamol may contribute to genotoxicity in humans.

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.

High doses of acetaminophen (400 mg/kg) or hydroxyurea (200 mg/kg) given intraperitoneally daily for 5 d caused reduction in relative testicular weight in mice (B6C3/F1/BOM M). Testicular atrophy of several tubules was seen in the hydroxyurea-treated mice 5 d after the last exposure, whereas acetaminophen did not lead to such changes. Exposure to acetaminophen caused neither a depletion of glutathione in the testis nor a marked increase in covalent binding. In contrast, significant decreases in the incorporation of thymidine into the testis were observed during the first 3 h following a single treatment with acetaminophen (100 to 400 mg/kg) or hydroxyurea (100 to 200 mg/kg). In mice treated with acetaminophen (400 mg/kg) or hydroxyurea (200 mg/kg) daily for 5 d, flow cytometric analysis revealed large reductions in one of the tetraploid populations of testicular cells (mostly early pachytene spermatocytes) on days 5 and 10. Changes in the populations of the various spermatid stages occurred later; thus, both compounds appeared to cause a delay in spermiogenesis. Indications of abnormal chromatin structure were seen in an increased frequency of vas deferens sperm on days 27 and 33 after the last exposure, when measured as increased susceptibility towards DNA denaturation in situ. In conclusion, high doses of acetaminophen or hydroxyurea inhibit DNA synthesis in the testis. The present data indicate that this leads to reduced testicular weight, a reduction in the number of early pachytene spermatocytes, changes in the proportions of the various spermatid stages, and an apparent alteration in sperm chromatin structure.

Biological pollution of indoor air can consist of viruses, bacteria, fungi, fungal spores, mites, dander and metabolic products from the microorganisms. The occurrence of such pollution can occasionally cause building-related adverse health effects. Allergens produced by house dust mites and in the dander of furred domestic animals are generally regarded to be among the most important causes of disease episodes in atopic individuals. Bacteria and fungi as such cause building-related disease more rarely. An exception is respiratory infections caused by Legionella bacteria. Other possible health effects of biological indoor air pollution have been suggested, but the data are inconclusive.

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.

The genotoxicity of paracetamol, including covalent binding to DNA, induction of DNA single-strand breaks (SSBs), and inhibition of replicative and repair synthesis of DNA, has been investigated in rodents in vivo. In the covalent binding studies male ICR mice were fasted and pretreated with diethyl maleate to deplete hepatic glutathione (GSH) and 300 mg/kg of [G-3H]paracetamol was administered intraperitoneally (i.p.). Animals were killed at 2, 6, 24, 72 and 168 h after paracetamol and hepatic or renal DNA and protein were isolated and the extent of covalent binding determined. Maximal binding to liver DNA, 8.4 +/- 3.1 pmol/mg of DNA, was observed at 2 h and declined rapidly to 2.6 pmol/mg at 24 h. Measurable binding (1.4 pmol/mg of DNA) was detected at 7 days. Protein binding in the liver in these animals peaked between 2 and 6 h (887 pmol/mg of protein at 2 h) and declined monoexponentially to 52 pmol/mg at 7 days. Although based on a limited body of data, covalent binding was also detected in DNA isolated from the kidney. DNA damage measured as SSBs by alkaline elution was induced in nuclear DNA isolated from the liver but not from the kidney, 2 h after i.p. injection of paracetamol at 600 mg/kg in male B6 mice. Only marginal DNA damage was noted at 300 mg/kg. The alkaline elution profile from damaged liver nuclei was markedly biphasic, suggesting that breaks were induced in DNA from a subpopulation of liver cells. The non-hepatotoxic paracetamol regioisomer, acetyl-m-aminophenol (600 mg/kg), which binds covalently to proteins, did not cause DNA SSBs.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of paracetamol on the repair of DNA damage in resting human peripheral mononuclear blood cells (MNC) in vitro were investigated by means of the alkaline elution technique. Low doses of UV light (254 nm, 3 J/m2) caused a transient increase in the amount of DNA single-strand breaks and alkali-labile sites (SSBs). Paracetamol (0.1-1.0 mM) present during post-irradiation incubation approximately doubled the maximum level of UV-induced (1-3 J/m2) SSBs and delayed the completion of repair. Although there were considerable variations between cells prepared from different donors, the level of UV-induced DNA SSBs was always higher with paracetamol. Hydroxyurea (0.3 mM), an inhibitor of ribonucleotide reductase, caused a similar increased accumulation and slow removal of SSBs, whereas cytosine-1-beta-D-arabinofuranoside (Ara C) (10 microM), an inhibitor of DNA polymerases, led to a steady accumulation of DNA SSBs. The increased levels of SSBs caused by paracetamol or hydroxyurea were both completely suppressed by concomitant addition of deoxyribonucleosides; this supports the notion that paracetamol as well as hydroxyurea inhibits ribonucleotide reductase. About the same rates of formation and removal of UV-induced SSBs were observed in T lymphocytes, B lymphocytes and monocytes. In both isolated T lymphocytes and B lymphocytes, paracetamol (0.3 mM) markedly increased the level of DNA SSBs induced by UV, whereas monocytes seemed to be less sensitive to the effect of paracetamol. It is concluded that the inhibition of DNA repair may contribute to the clastogenic effects of paracetamol.

Paracetamol was given to 10 healthy human volunteers in 3 doses of 1 g each during a period of 8 h. Blood samples for lymphocyte cultures were taken before and 24 h after paracetamol administration. A small but significant increase was found in the frequency of sister-chromatid exchanges (SCE) after intake of paracetamol (0.187 +/- 0.030 per chromosome before and 0.208 +/- 0.024 per chromosome after). After exposure the mean frequency of chromatid breaks per 100 cells was significantly increased (2.16 +/- 1.33 versus 0.33 +/- 0.50 before exposure). Exposure of human lymphocytes in vitro showed that concentrations of paracetamol above 0.1 mM induced inhibition of replicative DNA synthesis. Increased SCE was found in lymphocytes exposed to 1-10 mM paracetamol for 2 h. Furthermore, 0.75-1.5 mM paracetamol exposure for 24 h increased the frequency of chromatid and chromosome breaks in the lymphocytes. The paracetamol-induced SCE and chromosome aberrations may be secondary effects of paracetamol-induced inhibition of DNA synthesis or due to covalent binding of paracetamol metabolite(s) to DNA.

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.

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.

Experimental and theoretical evidence pertaining to cytotoxic and genotoxic activity of paracetamol in biological systems was used to formulate a simple mechanistic hypothesis to explain the relative inhibition of replicative DNA synthesis by a series of 19 structurally similar paracetamol analogues, 5 of which were specifically analyzed for the current study. It was hypothesized that the observed activity variation of the paracetamol analogues was based on the relative abilities of these compounds to undergo H atom loss at the phenolic oxygen, and on the relative stabilities of the resulting free-radical species. Three calculated parameters were found to be relevant--the partial atomic charge on the ring carbon attached to the phenolic oxygen, the partial charge on the phenoxy radical oxygen, and the energy difference between the parent phenolic paracetamol analogue and the corresponding radical dissociation products. The variation in parameter values was significantly correlated with the relative inhibition of DNA synthesis and was easily rationalized in terms of the mechanistic hypothesis proposed. More specifically, competitive reaction with a tyrosyl radical species involving the transfer of a hydrogen atom at the active site of ribonucleotide reductase was suggested as the underlying mechanistic basis for the observed activity variation of the paracetamol analogues. Comparison of calculated parameters for a model tyrosyl species and the paracetamol analogues was entirely consistent with this view.

1,2-Dibromo-3-chloropropane (DBCP) induced DNA damage, measured by alkaline elution, at low concentrations (5-10 microM) in suspensions of hepatocytes and testicular cells isolated from rats. At higher concentrations (greater than or equal to 100 microM) DBCP caused DNA damage and increased the frequency of sister chromatid exchanges in Chinese hamster V79 cells. When DBCP (2.5-10 microM) was tested for its ability to cause unscheduled DNA synthesis (UDS) in monolayers of liver cells isolated from untreated rats, a clear positive response was obtained. No increase in UDS was detectable when liver cells isolated from PCB-pretreated rats were used. In contrast, DBCP (greater than or equal to 50 microM) was metabolized in hepatocytes from PCB-pretreated rats to products mutagenic to Salmonella typhimurium TA100 in co-culture with the hepatocytes, whereas control hepatocytes were substantially (40-fold) less active. No bacterial mutagenicity could be detected when the TA100 strain was co-incubated with isolated rat testicular cells and DBCP. In co-cultures of hepatocytes and V79 cells DBCP-induced DNA damage in V79 cells occurred at low concentrations (10 microM DBCP) compared to the concentration (100 microM) needed to induce DNA damage in the V79 cells incubated without hepatocytes. Testicular cells were not able to enhance DBCP-induced DNA damage in the V79 cells. The data indicate that the putative reactive DBCP episulphonium ion metabolite formed in the testicular cells presumably responsible for testicular cell DNA damage, is not capable of escaping the cell where it is formed. Other reactive DBCP metabolites generated in the liver cells seem to be able to interact with the DNA of neighbouring cells.

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)

N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride (procarbazine; 50-1000 micrograms/ml) induced DNA damage in hepatocytes measured by an automated alkaline elution method, whereas no significant increase in unscheduled DNA synthesis was seen. In hepatocytes isolated from PCB-treated rats, DNA damage was detected in both test systems at concentrations as low as 1-10 micrograms/ml. DNA damage, as measured by alkaline elution and sister-chromatid exchange(s), was observed also in V79 cells incubated with PCB-hepatocytes. In contrast, no mutagenic activity was observed in the Salmonella typhimurium strain TA1530 co-incubated with the hepatocytes. Exposure of rats to low doses of procarbazine (25-50 mg/kg) caused DNA damage measured by alkaline elution in liver and testis, with the liver being somewhat more sensitive. The genotoxicity caused by procarbazine was increased by a factor of 2-3 in both organs by PCB-treatment of the rats. N-isopropyl-alpha-(2-methyl-hydrazino)-p-[alpha,alpha-2H2]toluamide (d2-procarbazine), was found to cause significantly less genotoxicity in control rats than either procarbazine itself, or N-isopropyl-alpha-(2-[alpha,alpha,alpha-2H3]methylhydrazino)-p-tol uamide (d3-procarbazine). This indicates that benzylic C-H oxidation of procarbazine is an important step in the activation of procarbazine to genotoxic metabolites in uninduced rats.

Cells cultured in vitro can be adapted to grow in the presence of high concentrations of fluoride by stepwise increasing the fluoride concentration in the culture medium. Such cells seem to have the ability to exclude fluoride from intracellular compartments.

Tris(2,3-dibromopropyl)phosphate (Tris-BP) was activated to mutagens in the Salmonella/microsome quantitative test system. Liver microsomes from rats pretreated with phenobarbital (PB) increased the mutagenicity of 0.05 mM Tris-BP to 186% of the activity obtained with liver microsomes from untreated rats. The addition of 0.02 mM Tris-BP to V79 Chinese hamster cells co-incubated with liver microsomes from PB-pretreated rats increased the number of mutants by a factor of 9.7. Tris-BP also caused genotoxic and cytotoxic responses in primary monolayers of rat hepatocytes. The relative increase in unscheduled DNA synthesis after treatment with 0.05 mM Tris-BP was 2.3-fold as measured by scintillation counting of radiolabelled thymidine incorporated into DNA of isolated nuclei. The use of hepatocytes isolated from PB-pretreated rats reduced the increases in DNA repair synthesis relatively to that in control cells. Monolayers of hepatocytes from untreated rats co-cultured with Salmonella typhimurium TA100 activated Tris-BP to mutagenic intermediates which were released into the culture medium. The studies with the V79 and liver-cell systems indicate that the reactive intermediates formed from Tris-BP are sufficiently stable and lipophilic to traverse the various membranes from the site of generation to the respective cellular targets. The relative degree of genotoxic responses of bis(2,3-dibromopropyl)phosphate, 2,3-dibromopropylphosphate, tris(2,3-bromopropyl)phosphate, tris(2-bromopropyl)phosphate and 2,3-dibromopropanol in the systems studied did not indicate that these compounds were proximate or ultimate reactive metabolites of Tris-BP in liver-derived activation systems.

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 of ODC-activity, fluoride had no effect on the cellular polyamine content during the experimental period (10 hours).

The established cell line LS (mouse fibroblasts) has been adapted to grow at otherwise toxic concentrations of fluoride. Different substrains of adapted LS cells consumed less glucose and converted less of the glucose to lactate than the unadapted cells. Differences were also found in the two parameters dependent on the number of generations grown in the presence of fluoride. The more generations with fluoride, the less lactate was produced and the less glucose consumed.

The genotoxic potential of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and their N-acetylated metabolites (AcIQ and AcMeIQ, respectively) has been studied, in order to evaluate whether an initial N-acetylation of IQ or MeIQ is important for the overall in vivo genotoxicity of the compounds. When incubated with uninduced (control) rat hepatocytes, both the acetylated and the unacetylated compounds appeared to be relatively stable, whereas water-soluble metabolites (i.e. not extractable by ethyl acetate at alkaline pH) were rapidly formed with hepatocytes from PCB-induced animals. No DNA damage was induced by IQ or MeIQ in hepatocytes isolated from control rats, as measured by alkaline elution. In hepatocytes from PCB-pretreated rats, IQ, MeIQ, AcIQ and AcMeIQ induced DNA damage at low (10(-6) M) concentrations, with AcIQ being more potent than IQ whereas AcMeIQ was less potent than MeIQ. Similar patterns were observed when unscheduled DNA synthesis was measured in hepatocytes. The compounds induced sister chromatid exchanges in Chinese hamster V79 cells with PCB-induced hepatocytes as activation system; IQ and AcIQ were equal while AcMeIQ had less activity than MeIQ. The compounds were also compared in bacterial mutagenesis test systems (Salmonella typhimurium TA98). With hepatocyte activation, AcIQ was slightly more potent than IQ, whereas AcMeIQ was markedly less mutagenic than MeIQ. With subcellular fractions as activation system (rat liver S9 or microsomes), the N-acetylated compounds were similar to or less mutagenic than their parent compounds. The mutagenic effects of AcIQ and AcMeIQ in bacteria with microsomal activation were markedly reduced by the deacetylase inhibitor paraoxon.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Low concentrations of paracetamol (0.1 mm) inhibited DNA synthesis in V79 Chinese hamster cells within minutes of addition. RNA and protein synthesis were inhibited only after longer exposure (3 hr) to considerably higher concentrations of paracetamol (3-10 mm). Flow-cytometry studies showed that V79 cells exposed to paracetamol were blocked initially in the G(1)-phase and subsequently slowly passed through the S-phase. No metabolites were detected by high-performance liquid chromatography after exposure of V79 cells to paracetamol for 50 hr. Furthermore, metyrapon and SKF 525-A (inhibitors of cytochrome P-450), indomethacine (inhibitor of prostaglandin H synthetase), and paraoxon (inhibitor of deacetylase) did not prevent paracetamol-induced inhibition of DNA synthesis. On the other hand, low concentrations of ascorbate (2 mum) reduced the effect of paracetamol. No effect was observed with the non-reductive precursor of ascorbate, gulonolactone. Neither catalase nor superoxide dismutase reduced the effect of paracetamol on DNA synthesis. Ascorbate also partly reduced paracetamol-induced sister-chromatid exchange (SCE). The inhibition of DNA synthesis caused by hydroxyurea, an inhibitor of ribonucleotide reductase, was also counteracted by ascorbate. Both paracetamol and hydroxyurea caused increased frequencies of SCE in V79 cells. We suggest that a specific enzyme involved in the synthesis of DNA is inactivated by the oxidation of paracetamol.

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.

Four endodontic sealer materials and some of their chemical constituents were subjected to Ames' test for mutagenicity with the Salmonella/microsome assay. Extracts of two zinc oxide-eugenol based materials and of one gutta-percha-zinc oxide-chloroform based sealer were negative in the test. Extracts of a synthetic polymer material, based on epoxy-bis-phenol A, induced mutations in Salmonella typhimurium TA 100 as did extracts of the epoxy-bis-phenol A resin alone. Formaldehyde, an active ingredient from one of the ZnO-based materials, induced mutations in both Salmonella typhimurium TA 98 and TA 100. The mutagenic activity of formaldehyde as well as of the epoxy material was reduced in the presence of rat liver microsomes.

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.

The uptake of spermine and spermidine in human heart cells in culture has been studied. Spermidine was accumulated to a higher degree than spermine whereas more spermine was absorbed to the cell surface. The results indicate that the uptake of spermidine and spermine is mediated by a common carrier. In addition, spermine enters the cells by several other transport mechanisms. The accumulated polyamines were not removed by washing with spermine or spermidine solutions, but they were released by treatment of the cells with butanol.

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.

The fluoride sensitivity, determined as effect on protein synthesis (incorporation of 14C-leucine), of liver and kidney cells in suspension culture was exposed. The cells were freshly prepared by collagenase perfusion from rats given drinking water with or without addition of 100 p.p.m. (5.26 mM) fluoride for 9-28 weeks. The fluoride sensitivity of the liver cells from rats given fluoride sensitivity of the kidney cells from fluoride exposed and control rats appeared similar. Fluoride resistance (i.e. decreased sensitivity) may thus develop also in cells in vivo. When exposed to 3 mM NaF for 1 hour the intracellular concentration of fluoride in liver cells from fluoride exposed and controls animals were similar.

Long-term (several years) exposure to fluoride induced decreased production of lactate in cultured, fluoride resistant LS cells. In intact, sensitive cells, 6 mM NaF had no effect on glycolysis, whereas in homogenates, from both resistant and sensitive cells, lactate production was blocked by 6 mM NaF, indicating the cell membrane to be a barrier to fluoride. A lower intracellular rather than extracellular fluoride concentration was found in the sensitive cell with a ratio of 0.4, whereas fluoride resistant cells excluded fluoride from their intracellular milieu.

Exposure of V79 Chinese hamster cells to non-cytotoxic concentrations of paracetamol (4-hydroxyacetanilide, 4-HAA) increased sister chromatid exchange (SCE) in the absence of an external activation system. Furthermore, a selective inhibition of DNA synthesis was observed at low 4-HAA concentrations. The inhibition could be counteracted by the addition of ascorbate, indicating that the effect is caused by an oxidation product of 4-HAA. In attempt to clarify possible relationships between cytotoxicity, inhibition of DNA synthesis and increased SCE, we studied the effect of 4-HAA and some related structures on these parameters. The relative position of the amino group and the hydroxyl group on the aromatic ring appear to be important for the inhibition of DNA synthesis. Removal of either of the two groups, N-acetylation and/or alkylation of the aromatic ring or phenolic oxygen decreased the effect of the aromatic amine on DNA synthesis. A significant response on SCE was observed with 4-amino-phenol, 4-HAA, 2-HAA, 3,5-dimethyl-4-HAA, 3-HAA and 2,6-dimethyl-4-HAA (none of the other compounds were tested). The increase in SCE frequency caused by 4-HAA and its analogs does not seem to be related to more general cytotoxic effects. The relative potencies of the compounds for SCE induction paralleled, for the most part, their effects on DNA synthesis. However, the induction of SCE and the inhibition of DNA synthesis did not occur at comparable concentrations. Thus, the possibility that 4-HAA increases the frequency of SCE through some other mechanism cannot be excluded.

Fluoride decreased the initial uptake of 2-aminoisobutyric acid (AIB) in LS cells only in high (supralethal) concentrations and after several hours of exposure. The steady state level of AIB seemed somewhat more sensitive as it was halved after 23 hours exposure to 6 mM-NaF. The cellular levels of some natural amino acids were also reduced by fluoride. However, cells resistant to fluoride showed equally lowered levels of amino acids, indicating that the observed effect of fluoride on cellular amino acid levels may be of little importance.

The food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) were studied for their genotoxic potential using hepatocytes isolated from untreated and Aroclor 1254 (PCB) pretreated rats as an activation system. Monolayers of hepatocytes co-incubated with Salmonella typhimurium TA98 activated IQ and MeIQ to bacterial mutagens, with MeIQ being about twice as potent as IQ. The mutagenic activities of IQ and MeIQ were increased by using hepatocytes from PCB-pretreated rats. IQ and MeIQ also caused primary DNA damage in the hepatocytes as determined by increases in the rate of alkaline elution of DNA, as well as increases in DNA-repair synthesis. Furthermore, exposure of V79 cells co-cultured with PCB-pretreated hepatocytes to IQ and MeIQ showed evidence of increased sister-chromatid exchanges and a low and variable increase in the number of 6-thioguanine-resistant mutants. The genotoxic potency of IQ and MeIQ in mammalian cells was low or virtually absent compared to their extreme potency in bacteria. This could be due to a lower capacity of mammalian cells to further metabolize the so-called directly acting bacterial mutagens, formed by a cytochrome P-450 dependent N-hydroxylation, to their ultimate reactive forms.

Organic extracts of emissions from wood combustion have been fractionated by high performance liquid chromatography (HPLC) into 25-28 fractions. Each fraction was tested for mutagenic activity in a modified Ames Salmonella/microsome bioassay requiring one-third of the test volumes needed for the ususal test. Direct mutagenic activity was noted predominantly in the most polar fractions, whereas indirect mutagenic activity was associated with the fractions containing polycyclic aromatic hydrocarbons (PAH) and with polar fractions probably consisting of aza-arenes and aromatic amines.

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.