Metal tolerance of a range of birch clones (Betula pendula and Betula pubescens) originating from metal-contaminated sites in England, Wales, Belgium and Finland were tested in soils supplemented with several concentrations of copper (Cu) or zinc (Zn) (500, 2000, 5000 mg kg(-1) dry wt. soil of CuSO4. 5H(2)O or ZnSO4. 7H(2)O) for 4 months and with sub-toxic metal supplements (500 mg CuSO4, 2000 mg ZnSO4) for 6 months. When grown at high concentrations of metals, severe toxicity symptoms (growth inhibition, chlorosis, necrosis) and clear evidence for differences in tolerance to this toxicity were found in a subset of the clones. When all clones were grown at a much lower, sub-toxic level of metal, again significant differences could be found between some of the clones. Clones derived from the same population varied greatly in their tolerance. However, the overall pattern of metal specificity varied in agree ment with the type of soil contamination at the site of origin. The growth of the clones from Harjavalta Cu/nickel smelter area was 19% better in Cu than in Zn-supplemented soil, on average. The growth of clones from Maatheide Zn smelter are was 19% poorer in Cu- than in Zn-supplemented soil. Sensitive clones accumulated more Cu and Zn to the above-ground parts. Some birch clones were able to survive at about 20-fold higher than typical total background Cu or Zn concentrations, whereas most clones were able to grow without serious toxic symptoms at about 10-fold concentrations. (C) 2001 Elsevier Science Ltd. All rights reserved.

INRA, Bordeaux Aquitaine Res Ctr, Agron Unit, F-33883 Villenave Dornon, France. Limburgs Univ Ctr, Dept SBG, B-3590 Diepenbeek, Belgium. Univ Kuopio, Dept Ecol & Environm Sci, FIN-70211 Kuopio, Finland. Univ Tras os Montes & Alto Douro, P-5001 Villa Real, Portugal.Boisson, J, INRA, Bordeaux Aquitaine Res Ctr, Agron Unit, BP 81, F-33883 Villenave Dornon, France.

The aim of this study was to investigate whether an in vitro test can give an indication of the overall toxicity of fabric extracts, and whether this toxicity correlates with the toxicity of the dyes and finishes used. Thirteen textile dyes and dyed/finished cotton fabrics were tested by using the Hepa-1 cytotoxicity test. Black sulphur and two blue reactive dyes were the most toxic, with IC50 (the concentration at which the total protein content was 50% of the protein content of non-exposed cells) values of 40–65μg/ml. The least toxic dyes, the black and yellow mix reactive dyes, had IC50 values of 825μg/ml and 703μg/ml, respectively. There was no correlation between the toxicities of the dyes and the fabric extracts; the extract from naphtholdyed fabric was the most cytotoxic. These results strongly support the hypothesis that the toxicity of a fabric extract cannot be predicted directly from the toxicity of the dye itself. The results also showed that flame-retardant and water/soil-repellent finishes can alter the cytotoxicity. In vitro tests, as exemplified by the Hepa-1 cytotoxicity test, could provide useful information for developing new ecotextiles.

Three sulfur-containing compounds, 2,3,7,8-tetrachlorothianthrene (TCTA), 2,3,7,8-tetrachlorodibenzothiophene (TCDT), and 3,3,4,4'-tetrachlorodiphenyl sulfide (TCDPS), were analyzed for their CYP1A1-inducing potencies - measured as aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase (EROD) activities-in mouse hepatoma cell culture Hepa-1. Marked differences in the induction potencies were observed among the three compounds studied and between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its sulfur analogue. The estimated EC50 values for TCDD, TCTA, and TCDT were about 8 pM, 700 pM, and 7.5 nM, respectively. TCDPS did not elicit any AHH/EROD induction. Comparative molecular field analysis (CoMFA) was not able to predict correctly the biological potency of TCTA and TCDT. The most important reason for the poor performance of the model may be the positive point charge of sulfur in TCTA and TCDT.

Cytochrome P450IA1 (CYP1A1) induction of Hepa-1 mouse and H4IIE rat hepatoma cell lines was compared using selected environmental samples. The results were in agreement for both cell lines: no induction was observed for the fly ash extract from peat combustion, an intermediate induction was found for the fly ash extract from biosludge combustion, and a strong induction was detected for natural peat extract. However, Hepa-1 responded to the samples more sensitively than did H4IIE: the half maximal induction (ED50) values for Hepa-1 were smaller than those for H4IIE. In a bacterial DNA repair assay without metabolic activation and in a mammalian sister chromatid exchange test in the presence of metabolic activation the samples were virtually non-genotoxic. Thus the CYP1A1-inducing potency and genotoxicity of the samples were not correlated. In light of these results, the CYP1A1 induction test might be a useful addition to conventional genotoxicity tests, which may fail to detect potentially harmful compounds/mixtures.

Fly ash was collected from five large-scale or pilot tests in which burning of bleached kraft pulp mill sludge was studied. The content of dioxin-like compounds in this fly ash was estimated both chemically and biologically. Fly ash was analyzed chemically for 17 PCDD and PCDF congeners by high-resolution GC-MS, and the data were transformed to Nordic TCDD equivalents. The biological analyses were based on the induction of several enzymes (aryl hydrocarbon hydroxylase [AHH], 7-ethoxyresorufin 0-deethylase [EROD], aldehyde dehydrogenase-3 [ALDG3]) by the fly ash extracts in a mouse hepatoma cell line, Hepa-1. The inducing potencies were expressed as biological TCDD equivalents. There was a good correlation between the Nordic and the biological TCDD equivalents. Differences in the amounts of dioxin-like compounds among the combustions were attributed mainly to the boiler types and not to fuel characteristics or combustion parameters.

When the effects of the products of various combustion processes - with coal, heavy fuel oil, peat or hazardous waste as a fuel - were compared, an anomalous correlation between the biologically (induction of cytochrome P450IA1 in the mouse hepatoma cell line Hepa-1) and the chemically estimated ''TCDD equivalents'' was observed. The inducing effect of the product from a peat combustion operation was stronger than that of the product from hazardous waste combustion, although the chemically estimated TCDD equivalents were clearly lower in the former one. A more detailed study of two different peat combustion processes showed that induction in one of the combustion processes was due to the ''leakage'' of unburned peat to the fly ash. In the other process, no leakage and no cytochrome P450IA1 induction was evident. This led us to a more detailed study of peat itself. One fraction of the peat extract was shown to be a very potent inducer of cytochrome P45OIA1. However, only small amounts of polychlorinated dibenzodioxins (PCDDs) or furans (PCDFs) were present in this fraction. It is thus evident that there are cytochrome P450IA1 inducers in peat other than those revealed by the PCDD/PCDF analysis.

Induction of aryl hydrocarbon hydroxylase (AHH) activity in a mouse (Hepa-1) and a rat (H4IIE) hepatoma cell line was used as an indicator of biological effect of two coplanar polychlorinated biphenyls of different types. The previously uncharacterized pentachlorinated biphenyls used were non-ortho substituted 3,3',4,5,5'-PeCB (PCB 127) and mono-ortho substituted 2,3,3',4,5'-PeCB (PCB 108). In Hepa-1 cells, 50-mu-M 3,3',4,5,5'-PeCB caused nearly the same induction of AHH as 1 nM 2,3,7,8-TCDD (TCDD). It should be noted, however, that there was a 50,000-fold difference in the effective concentrations. 2,3,3',4,5'-PeCB was a much weaker inducer of AHH. As 50-mu-M concentration 2,3,3',4,5'-PeCB was cytotoxic to Hepa-1 cells, whereas 3,3',4,5,5'-PeCB was not. This supports some earlier observations that AHH induction and general cytotoxicity do not always coincide. In H4IIE, neither of the PeCBs was cytotoxic in the concentrations studied. However, both PeCBs were AHH inducers. The different response of the two cell lines studied implies that for the estimation of "TCDD equivalents" more than one cell line should be studied. According to ED50-values (about 10 pM for Hepa-1; about 15 pM for H4IIE) obtained from the dose-response curves, Hepa-1 was somewhat more responsive than H4IIE to TCDD.

The inducibility of cytochrome P450IA1 (detected as aryl hydrocarbon hydroxylase AHH and 7-ethoxyresorufin O-deethylase EROD) in the mouse hepatoma cell line Hepa-1 has been used as a bioassay for polycyclic organic compounds in fly ash samples collected from combustion of barking material, biosludge and natural gas in a fluidized bed combustor.An amount corresponding to 25 mg fly ash resulted in half of the maximal induction (ED50) of both AHH and EROD. The induction was only observed using the fly ash fraction, which contained planar aromatic compounds (PAHs, PCDDs and PCDFs, the so-called "TCDD-equivalents"). The fly ash extracts were not cytotoxic in the concentrations studied, as judged by counting viable cells or by determination of total protein content in the cultures.The Hepa-1 test, which measures induction of the cytochrome P4501A1 enzyme, seems to be a useful short-term bioassay when information about the biological response of complex environmental samples is needed.

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are ubiquitous in the environment. Combustion of organic material, particularly in the presence of chlorine, is a significant source of these harmful compounds. In this study, the induction of cytochrome P450IA1 - measured as aryl hydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities - in the mouse hepatoma cell line, Hepa-1, was used as a biological indicator of the presence of PCDDs and PCDFs in fly ash collected from combustion of chlorine containing material. The highest chlorine content in the combustions studied was 1.2 % (w/w) in a mixture of coal and bark. An amount corresponding to about 7.0 mg of the respective fly ash per ml of growth medium was enough to elicit a half-maximal (ED50) induction of cytochrome P450IA1 in Hepa-1 cells. The inducing compounds were located in the fly ash fraction which contained the PCDDs and PCDFs. There was a good correlation between the PCDD/PCDF composition of the fly ashes and the enzyme inducing effect they had.