Photolysis primes biodegradation of benzo[a]pyrene

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.     

Antimutagenic properties of a polyphenol-enriched extract derived from sesame-seed perisperm

A polyphenolic mixture derived from sesame-seed perisperm (SSP) strongly reduced the mutagenicity of hydrogen peroxide (H(2)O(2)), sodium azide (NaN(3)), and benzo[a]pyrene (BaP) in strains TA100 and/or TA98 of Salmonella typhimurium. It exhibited desmutagenic activity against H(2)O(2), BaP in TA98 and/or TA100 and biomutagenic activity (apparently by affecting the DNA-repair system) against NaN(3) in strain TA100. According to in vitro experiments the polyphenolic mixture inhibited the activity of the CYP1A1 (EROD) enzyme responsible for the activation of BaP in the Ames' test, as well as that of the cytosolic enzyme GST. A cytosolic fraction from liver of male Wistar rats treated with either 20% SSP in the food, or 3mg or 6 mg of polyphenolic mixture/20 g food/day for a time period of 8 weeks reduced the mutagenic potential of BaP in strains TA100 and TA98, with the cytosolic fraction from rats treated with SSP causing the strongest reduction. Furthermore, a microsomal fraction from the 20% SSP-treated rats inhibited the mutagenicity of BaP in strains TA100 (26.3%) and TA98 (23%). In contrast, a microsomal fraction from rats treated with 3mg of polyphenolic mixture stimulated the mutagenicity of BaP in TA100 but reduced it in TA98, while for the microsomal fraction from rats treated with 6 mg of polyphenolic mixture, these effects on TA100 and TA98 were reversed.     

Correlations between 15 polycyclic aromatic hydrocarbons (PAH) and the mutagenicity of the total PAH fraction in ambient air particles in La Spezia (Italy)

Airborne particulate matter has been monitored 4 times a month for 1 year (1988) in the city of La Spezia (Italy). The polycyclic aromatic hydrocarbon (PAH) fractions were extracted, purified and characterized for the content of 15 individual PAH. In general when concentrations of individual PAH were compared statistical correlation was obtained. Mutagenicity studies were performed by the use of the Ames plate test with the Salmonella strains TA98, TA100, TA98NR and TA98DNP6 with and without metabolic activation (S9 mix). The TA98 strain was by far the most responsive and the S9 mix was absolutely required as expected when PAH are assayed. Besides mutagenicity, toxicity was also considered and it proved to be correlated with mutagenicity in TA98, +S9. The TA98NR and TA98DNP6 strains showed no appreciable differences from the parental strain TA98 indicating the absence of significant amounts of direct-acting nitro derivatives in our PAH samples. Of the 15 PAH considered in this study the amounts of cyclopental[c,d]pyrene (CPP) correlated best with mutagenicity. The role of CPP in contributing to the indirect mutagenicity of urban air PAH samples is discussed.     

Antimutagenic properties of a polyphenol-enriched extract derived from sesame-seed perisperm

A polyphenolic mixture derived from sesame-seed perisperm (SSP) strongly reduced the mutagenicity of hydrogen peroxide (H₂O₂), sodium azide (NaN₃), and benzo[a]pyrene (BaP) in strains TA100 and/or TA98 of Salmonella typhimurium. It exhibited desmutagenic activity against H₂O₂, BaP in TA98 and/or TA100 and biomutagenic activity (apparently by affecting the DNA-repair system) against NaN₃ in strain TA100. According to in vitro experiments the polyphenolic mixture inhibited the activity of the CYP1A1 (EROD) enzyme responsible for the activation of BaP in the Ames’ test, as well as that of the cytosolic enzyme GST.A cytosolic fraction from liver of male Wistar rats treated with either 20% SSP in the food, or 3 mg or 6 mg of polyphenolic mixture/20 g food/day for a time period of 8 weeks reduced the mutagenic potential of BaP in strains TA100 and TA98, with the cytosolic fraction from rats treated with SSP causing the strongest reduction. Furthermore, a microsomal fraction from the 20% SSP-treated rats inhibited the mutagenicity of BaP in strains TA100 (26.3%) and TA98 (23%). In contrast, a microsomal fraction from rats treated with 3 mg of polyphenolic mixture stimulated the mutagenicity of BaP in TA100 but reduced it in TA98, while for the microsomal fraction from rats treated with 6 mg of polyphenolic mixture, these effects on TA100 and TA98 were reversed.     

Peroxidative activation of 3,3'-dichlorobenzidine to mutagenic products in the Salmonella typhimurium test

The direct and H2O2-dependent mutagenicity of 3,3'-dichlorobenzidine (DCB) were compared in Salmonella tester strains TA98, TA98/1,8-DNP6, TA100 and TA102 using the Ames test. DCB exhibited both direct and H2O2-dependent mutagenicity to both tester strains TA98 and TA98/1,8-DNP6. This H2O2-dependent mutagenicity of DCB was prevented by horseradish peroxidase. DCB, in contrast to its effects in tester strains TA98, was not mutagenic to TA100 and TA102 either directly or in the presence of H2O2. These results suggest that mechanisms, perhaps enzymes endogenous to tester strains TA98, may play a role in the activation of DCB.     

Selenium modified mutagenicity and metabolism of benzo[a]pyrene in an S9-dependent system

Selenium added to the incubation mix containing rat-liver S9 modified both the metabolism and mutagenicity of benzo[a]pyrene (BaP) and several of its metabolites. Selenium (Na2SeO3) inhibited the S9-dependent mutagenic effects of BaP on Salmonella typhimurium strain TA100 as indicated by the number of histidine-dependent revertants counted. This inhibition was concentration-dependent over a range of 12.5 to 100 ppm. When used as the substrate the BaP metabolites 7,8-dihydrodiol, 9,10-dihydrodiol and 3-hydroxy also produced significantly fewer revertants in TA100 when selenium was included in the incubation mix. High-performance liquid chromatographic analysis of metabolites from S9-dependent metabolism of BaP indicated that selenium inhibited the formation of 3-hydroxy-BaP, 9,10-dihydrodiol, 7,8-dihydrodiol, 1,3- and 3,6-quinone. Eluting samples on an alumina column to isolate the conjugated metabolites showed that selenium caused 12% less binding to glucuronides, no significant differences in binding to sulfate esters or glutathione but the amount of unmetabolized BaP and unconjugated metabolites was increased by 48%. These results suggest that selenium inhibits S9-dependent BaP metabolism therefore reducing the mutagenic effects of this compound.     

The influence of automobile exhausts on mutagenicity of soils: contamination with, fractionation, separation, and preliminary identification of mutagens in the Salmonella/reversion assay and effects of solvent fractions on the sister-chromatid exchanges in human lymphocyte cultures and in the in vivo mouse bone marrow micronucleus assay

To test the assumption that automobile exhausts contribute to soil mutagenicity, two soils with low levels of mutagenic activities were exposed to traffic exhausts at a heavily charged junction of German motorways (Autobahnen) for 3, 7, 10, 13, 17, 21, and 26 weeks. Indeed, in the presence of a metabolic activation system from rat liver (S9), an average increase of 8 and 9 (4 and 12) revertants per gram per week was found in Salmonella typhimurium TA 98 (TA 100). In the absence of S9, meaningful measurements were impossible on account of a concurrent dose dependent increase of toxicity. No correlation between the increase of mutagenicity and the contents of polycyclic aromatic hydrocarbons (PAH) could be detected. In another series, soils sampled at the roadside and at distances of 10 and 50m of five roads near Mainz expressed 10-20-fold higher mutagenicity (revertants per gram) under identical test conditions as compared with the average of agricultural soils. Toxic effects, however, again confounded the results and no correlation between the distance from roads and the levels of mutagenicity could be demonstrated. Subsequently, Soxhlet-extraction with the solvent sequence dichloromethane, acetone, and toluene/diethylketone was found to be an optimum procedure for soils at roadsides. The mass balance of solvent fractionation of such soils revealed that <2% each belonged to organic acids and bases, approximately 4% to fractions designed polar neutrals, approximately 8% to polar aromatics, approximately 7% to dichloromethane solubles, and approximately 79% to cylohexane solubles, among them approximately 63% acetone soluble compounds. The major part of mutagenicity (55-65%) was present in the fraction of polar aromatics, followed by polar neutrals and the acetone subfraction of cyclohexane solubles ( approximately 10% each) summarizing the results obtained with S. typhimurium TA 98, TA 98NR, YG 1021, YG 1024, TA 100, YG 1026, and YG 1029 with and without addition of S9. The modified tester strains, either deficient in nitroreductase (TA 98NR) or overproducing nitroreductase (YG 1021, 1026) or O-acetyl-transferase (YG 1024, 1026), indicated a major contribution of nitroarenes to soil mutagenicity. With respect to mutagenic PAH, high pressure liquid chromatography (HPLC) revealed that >90% of dibenz[a,h]anthracene (4.18mg/kg soil), benzo[a]pyrene (1.96mg), benzofluoranthenes (0.14mg), and benz[a]anthracene (0. 18mg) were present in the acetone subfraction of cyclohexane solubles. Concentrations and mutagenic activities, however, did not correlate. Additional preparative and analytical HPLC of the solvent fractions of polar neutrals and polar aromatics, resulted in the tentative identification of 2-nitrofluorene. Analysis of the vertical profile of soil revealed an increase of mutagenicity per gram from the surface to a maximum at 5-15cm depth and a subsequent decrease with very little activity remaining deeper than 35cm. In human lymphocyte cultures, the fraction of polar aromatics, 0.01-0. 3microg/ml, induced 11.27+/-4.76-20.70+/-6.19 sister-chromatid exchanges (SCE) per cell in the absence of S9 (solvent control: 10. 16+/-4.83 SCE per cell) and 12.77+/-6.53-17.87+/-4.93 SCE per cell in the presence of S9 (solvent control: 8.37+/-3.92 SCE per cell). However, no activities could be detected in the fractions of polar neutrals and non-polar neutrals. Again, negative results were obtained in the in vivo mouse bone marrow micronucleus assay at 2000mg/kg p.o. with all fractions.     

Antimutagenic activity of some naturally occurring compounds towards cigarette-smoke condensate and benzo[a]pyrene in the Salmonella/microsome assay

Several compounds, occurring in food, were tested for antimutagenic activity towards cigarette-smoke condensate (CSC) and benzo[a]pyrene (BaP). Antimutagenicity was determined in the Salmonella/microsome test, with tester strain TA98, in the presence of rat-liver homogenate. Dose-response curves did show reduction of CSC- and BaP-induced mutagenicity by ellagic acid, riboflavin and chlorophyllin. Chlorophyll a and chlorophyll b, although less distinct, also inhibited CSC- and BaP-induced mutagenicity. Ascorbic acid, beta-carotene, tocopherol acetate, chlorogenic acid and butyl hydroxyanisole did not have any influence on the mutagenicity of CSC and BaP. The similarity in results for cigarette-smoke condensate and for BaP indicates that a general mechanism may be involved in the inhibition of CSC- and BaP-induced mutagenicity.     

Polycyclic aromatic hydrocarbons and phthalic acid esters in the soil-radish (Raphanus sativus) system with sewage sludge and compost application

We studied the accumulation of polycyclic aromatic hydrocarbons (PAHs) and phthalic acid esters (PAEs) in a latosolic red soil and radish (Raphanus sativus) with application of sewage sludge at rates of 10, 20 and 40 g kg(-1) soil or compost at rate of 10 g kg(-1) soil. In radish the concentrations of individual PAHs and PAEs varied from non-detectable to 803 microg kg(-1) dry weight (d.w.) and from non-detectable to 2048 microg kg(-1) d.w., respectively. Compared to the control, higher application rates of sewage sludge resulted in pronounced increases in shoot, root and soil concentrations of PAHs and PAEs. PAE concentrations in radish grown in soil spiked with sludge compost were higher while the PAH concentrations were comparable to those receiving 10 g kg(-1) of sewage sludge. However, the root biomass of radish in soil amended with compost was significantly higher and the shoot-to-root ratio was significantly lower than in the other treatments. The bioconcentration factors (BCFs, the ratio of contaminant concentration in plant tissue to the soil concentration) of di-n-butyl phthalate and di(2-ethylhexyl) phthalate in both shoots and roots and of total PAH concentrations in roots were less than 1.0, but some BCFs for individual PAHs were high with a maximum value of 80.     

Mutagenicity and polycyclic aromatic hydrocarbons analysis of airborne particulate matters from Taipei City

The mutagenic activity of dichloromethane extracts of 147 air particulate samples collected from 8 stations during December 1986-June 1987 in Taipei city was consistently higher in S. typhimurium strain TA 98 than in strain TA 100 in the presence of S9 mixture. Among the 8 stations, Nan Kang Police Station, Fu Hsing Elementary School, and Chung Hsing University which were located in the industrial district, downtown area, and heavy traffic zone, respectively, had significantly higher levels of PAHs than the other stations. In contrast, the levels of PAHs were much lower in the suburban station, near Pei Tou Elementary School. However, PAH contents of the air particulate samples collected from these stations did not show good correlation with their mutagenicity. The air particulates collected at some stations on Sunday when the traffic changed from heavy to light showed lower mutagenicity and PAH contents as compared with the other weekdays at the same stations. On the contrary, the samples collected at Pei Tou station in a suburban area where the traffic changed from light to heavy on Sunday showed higher mutagenicity and PAH contents. The monthly average of PAHs of air particulate samples collected over a 7-month period from 8 stations in Taipei city was lower than the average in 1980. Moreover, when compared with other countries, such as U.S.A., the Netherlands, West Germany, Italy, Norway, and Japan, the levels of PAHs and mutagenicity of air particulate matters in Taipei city were similar or slightly lower. The mutagenicity and contents of PAHs of air particulates collected from burnt ABS were significantly higher than those of burnt PVC. One sample PT-6-3 was collected while a nearby garbage collection area was on fire. The mutagenicity of that sample increased 3 to 16 fold and contained an 11 to 33 times higher content of the six PAHs (BaP, BeP, BbF, BaA, Chr, and DbA) as compared with the other samples collected at the same location at a different time. The higher mutagenicity and PAH contents of that sample might be due to the pollution of the air from combustion of the garbage containing products made of ABS.     

Use of a Salmonella microsuspension bioassay to detect the mutagenicity of munitions compounds at low concentrations

Past production and handling of munitions has resulted in soil contamination at various military facilities. Depending on the concentrations present, these soils pose both a reactivity and toxicity hazard and the potential for groundwater contamination. Many munitions-related chemicals have been examined for mutagenicity in the Ames test, but because the metabolites may be present in low environmental concentrations, a more sensitive method is needed to elucidate the associated mutagenicity. RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), TNT (2,4,6-trinitrotoluene), tetryl (N-methyl-N-2,4,6-tetranitroaniline), TNB (1,3,5-trinitrobenzene) and metabolites were examined for mutagenicity in a microsuspension modification of the Salmonella histidine reversion assay with and without metabolic activation. TNB and tetryl were positive in TA98 (32.5, 5.2revertants/nmole) and TA100 (7.4, 9.5revertants/nmole) without metabolic activation and were more potent than TNT (TA98, 0.3revertants/nmole; TA100, 2.4revertants/nmole). With the exception of the tetranitroazoxytoluene derivatives, TNT metabolites were less mutagenic than TNT. RDX and two metabolites were negative in both strains, however, hexahydro-1,3,5-trinitroso-1,3,5-triazine was positive in TA100 with and without S9. Microsuspension bioassay results tend to correlate well with published Ames test data, however, there are discrepancies among the published data sets and the microsuspension assay results.     

The influence of automobile exhausts on mutagenicity of soils: contamination with, fractionation, separation, and preliminary identification of mutagens in the Salmonella/reversion assay and effects of solvent fractions on the sister-chromatid exchanges in human lymphocyte cultures and in the in vivo mouse bone marrow micronucleus assay

To test the assumption that automobile exhausts contribute to soil mutagenicity, two soils with low levels of mutagenic activities were exposed to traffic exhausts at a heavily charged junction of German motorways (Autobahnen) for 3, 7, 10, 13, 17, 21, and 26 weeks. Indeed, in the presence of a metabolic activation system from rat liver (S9), an average increase of 8 and 9 (4 and 12) revertants per gram per week was found in Salmonella typhimurium TA 98 (TA 100). In the absence of S9, meaningful measurements were impossible on account of a concurrent dose dependent increase of toxicity. No correlation between the increase of mutagenicity and the contents of polycyclic aromatic hydrocarbons (PAH) could be detected. In another series, soils sampled at the roadside and at distances of 10 and 50 m of five roads near Mainz expressed 10–20-fold higher mutagenicity (revertants per gram) under identical test conditions as compared with the average of agricultural soils. Toxic effects, however, again confounded the results and no correlation between the distance from roads and the levels of mutagenicity could be demonstrated. Subsequently, Soxhlet-extraction with the solvent sequence dichloromethane, acetone, and toluene/diethylketone was found to be an optimum procedure for soils at roadsides. The mass balance of solvent fractionation of such soils revealed that <2% each belonged to organic acids and bases, 4% to fractions designed polar neutrals, 8% to polar aromatics, 7% to dichloromethane solubles, and 79% to cylohexane solubles, among them 63% acetone soluble compounds. The major part of mutagenicity (55–65%) was present in the fraction of polar aromatics, followed by polar neutrals and the acetone subfraction of cyclohexane solubles (10% each) summarizing the results obtained with S. typhimurium TA 98, TA 98NR, YG 1021, YG 1024, TA 100, YG 1026, and YG 1029 with and without addition of S9. The modified tester strains, either deficient in nitroreductase (TA 98NR) or overproducing nitroreductase (YG 1021, 1026) or O-acetyl-transferase (YG 1024, 1026), indicated a major contribution of nitroarenes to soil mutagenicity. With respect to mutagenic PAH, high pressure liquid chromatography (HPLC) revealed that >90% of dibenz[a,h]anthracene (4.18 mg/kg soil), benzo[a]pyrene (1.96 mg), benzofluoranthenes (0.14 mg), and benz[a]anthracene (0.18 mg) were present in the acetone subfraction of cyclohexane solubles. Concentrations and mutagenic activities, however, did not correlate. Additional preparative and analytical HPLC of the solvent fractions of polar neutrals and polar aromatics, resulted in the tentative identification of 2-nitrofluorene. Analysis of the vertical profile of soil revealed an increase of mutagenicity per gram from the surface to a maximum at 5–15 cm depth and a subsequent decrease with very little activity remaining deeper than 35 cm. In human lymphocyte cultures, the fraction of polar aromatics, 0.01–0.3 μg/ml, induced 11.27±4.76–20.70±6.19 sister-chromatid exchanges (SCE) per cell in the absence of S9 (solvent control: 10.16±4.83 SCE per cell) and 12.77±6.53–17.87±4.93 SCE per cell in the presence of S9 (solvent control: 8.37±3.92 SCE per cell). However, no activities could be detected in the fractions of polar neutrals and non-polar neutrals. Again, negative results were obtained in the in vivo mouse bone marrow micronucleus assay at 2000 mg/kg p.o. with all fractions.     

Activation of anti-Trypanosoma cruzi drugs to genotoxic metabolites promoted by mammalian microsomal enzymes

Salmonella typhimurium TA100 and its nitroreductase-deficient derivative, TA100 NR, were used to reevaluate the mutagenic activities of benznidazole and nifurtimox. Mutagenicity and toxicity of nifurtimox were abolished in the TA100 NR tester strain under aerobic or anaerobic conditions and addition of rat liver extracts did not alter the results. However, benznidazole showed a significant mutagenicity and toxicity to the nitroreductase-deficient strain TA100 NR under hypoxic conditions. Addition of rat liver extracts enhanced the observed mutagenicity and toxicity of benznidazole even more. In the presence of O2 the genotoxic activities of benznidazole to the TA100 NR tester strain were eliminated. These results lead us to conclude that bacterial enzymes were responsible for the previously observed genotoxic effects of nifurtimox and benznidazole on S. typhimurium TA100. Moreover, under anaerobic conditions, only benznidazole could be metabolized by mammalian nitroreductases into a mutagenic derivative.     

Functioning and toxicity of artificial soil mixtures with metal-bearing sewage sludge

Various artificial soil mixtures were prepared by mixing two different toxic metals containing sewage sludge from Ljubljana and Maribor wastewater treatment plants with natural mineral soil. The plots with mixtures were exposed to field environmental conditions for a period of 1 year, after which we assessed soil toxicity (germination test with Lactuca sativa), potential metal phyto-accessibility (diethylenetriamine pentaacetic acid – DTPA extraction test), soil functioning (by soil enzymes activity) and conducted a field growth test with Lollium perenne L. as a metal bio-indicator plant. The metal phyto-accessibility extraction test (DTPA) showed lower values than the metal accumulation test with L. perenne L., which also showed higher metal concentrations in roots compared to leaves. With the exception of the mixture containing 30% (w/w) of sludge from the Ljubljana wastewater treatment plant, all mixtures containing more than 20% of sludge negatively affected root elongation of L. sativa seeds, indicating an increase in artificial soils toxicity. Increasing the ratio of sludge from the Ljubljana plant increased dehydrogenase and decreased phosphomonoesterase, while the addition of sludge from the Maribor plant increased phosphomonoesterase activity. Overall, the effect of sludge addition on artificial soil properties, toxicity and functioning not only depended on dosage but was also sensitive to the source and pre-treatment of the sewage sludge.     

Evaluation of a battery of Salmonella typhimurium tester strains for biomonitoring of mutagenic polycyclic aromatic hydrocarbons, nitroarenes and aromatic amines

Various combinations of Salmonella typhimurium tester strains and S9 mix for bioactivation (TA98+S9 mix, TA98S; YG1041+S9 mix, YG1041S) and strain YG1041 in the absence of S9 mix (YG1041) were used to evaluate the mutagenic activity of eight polycyclic aromatic hydrocarbons (PAHs), seven nitroarenes (NAs) and seven aromatic amines (AAs). Three cigarette smoke extracts and two extracts of smokers' urine (SUE) were also included. Urinary mutagenicity was then determined on 31 individuals, potentially exposed to PAHs, for 0 h, 7 h, 12 h and 24 h. Concentrations of urinary 1-hydroxypyrene (1OHP) and 3-hydroxybenzo[a]pyrene (3OHBaP), the levels of atmospheric pyrene (Py) and benzo[a]pyrene (BaP), and particulate concentrations in air (AP) were also measured. PAHs could be detected by TA98S and YG1041S, with TA98S being more sensitive than YG1041S. While NAs could be detected by all combinations, YG1041 and YG1041S were more sensitive than TA98S. Although both YG1041S and TA98S could detect AAs, YG1041S was more sensitive than TA98S. Cigarette smoke extract contained mutagenic AAs and NAs, but AAs were the only mutagenic compounds detected in the extracts of smokers' urine. The concentrations of 1OHP (7 h and 12 h) were significantly higher than those at 0 h, but no difference could be detected with 3OHBaP. Correlations were found between Py and 1OHP (7 h and 24 h) and between BaP and 3OHBaP concentrations (7 h, 12 h and 24 h). A significantly elevated urinary mutagenicity was detected with YG1041S at 7h in the group of smokers. A good correlation was determined between AP and the test results with TA98S (7 h) and with YG1041 (0 h and 7 h). Urinary 1OHP correlated with the test results with YG1041S (0 h, 7 h and 12 h) while 3OHBaP correlated with those obtained with YG1041S (7 h). Overall, 21/31 individuals were occupationally exposed to AAs, 15/31 individuals were exposed to NAs, and 2/31 were exposed to PAHs as indicated by the Salmonella mutagenicity assay. The urine mutagenicity test was not effective at monitoring occupational exposure to PAHs. However, the correlation with AP implied the presence of unknown mutagenic atmospheric substances that could modulate the urinary mutagenicity.     

Mutagens in contaminated soil: a review

The intentional and accidental discharges of toxic pollutants into the lithosphere results in soil contamination. In some cases (e.g., wood preserving wastes, coal-tar, airborne combustion by-products), the contaminated soil constitutes a genotoxic hazard. This work is a comprehensive review of published information on soil mutagenicity. In total, 1312 assessments of genotoxic activity from 118 works were examined. The majority of the assessments (37.6%) employed the Salmonella mutagenicity test with strains TA98 and/or TA100. An additional 37.6% of the assessments employed a variety of plant species (e.g., Tradescantia clone 4430, Vicia faba, Zea mays, Allium cepa) to assess mutagenic activity. The compiled data on Salmonella mutagenicity indicates significant differences (p<0.0001) in mean potency (revertents per gram dry weight) between industrial, urban, and rural/agricultural sites. Additional analyses showed significant empirical relationships between S9-activated TA98 mutagenicity and soil polycyclic aromatic hydrocarbon (PAH) concentration (r2=0.19 to 0.25, p<0.0001), and between direct-acting TA98 mutagenicity and soil dinitropyrene (DNP) concentration (r2=0.87, p<0.0001). The plant assay data revealed excellent response ranges and significant differences between heavily contaminated, industrial, rural/agricultural, and reference sites, for the anaphase aberration in Allium cepa (direct soil contact) and the waxy locus mutation assay in Zea mays (direct soil contact). The Tradescantia assays appeared to be less responsive, particularly for exposures to aqueous soil leachates. Additional data analyses showed empirical relationships between anaphase aberrations in Allium, or mutations in Arabidopsis, and the 137Cs contamination of soils. Induction of micronuclei in Tradescantia is significantly related to the soil concentration of several metals (e.g., Sb, Cu, Cr, As, Pb, Cd, Ni, Zn). Review of published remediation exercises showed effective removal of genotoxic petrochemical wastes within one year. Remediation of more refractory genotoxic material (e.g., explosives, creosote) frequently showed increases in mutagenic hazard that remained for extended periods. Despite substantial contamination and mutagenic hazards, the risk of adverse effect (e.g., mutation, cancer) in humans or terrestrial biota is difficult to quantify.     

Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic.

The mutagenic activities associated with inhalable airborne particulate matter (PM10) collected over a year in four towns (Czech Republic) have been determined. The dichloromethane extracts were tested for mutagenicity using the Ames plate incorporation test and the Kado microsuspension test both with Salmonella typhimurium TA98 and its derivative YG1041 tester strains in the presence and absence of S9 mixture. The aim of this study was to assess the suitability of both bacterial mutagenicity tests and to choose the appropriate indicator strain for monitoring purposes. To elucidate the correlation between mutagenicity and polycyclic aromatic hydrocarbons (PAHs), the concentration of PAHs in the air samples were determined by GC/MS. In general, the significant mutagenicity was obtained in organic extracts of all samples, but differences according to the method and tester strain used were observed. In both mutagenicity tests, the extractable organic mass (EOM) exhibited higher mutagenicity in the YG1041 strain (up to 97 rev/microg in the plate incorporation and 568 rev/microg in the microsuspension tests) than those in TA98 (up to 2.2 rev/microg in the plate incorporation and 14.5 rev/microg in the microsuspension tests). In the plate incorporation test, the direct mutagenic activity in YG1041 was on average 60-fold higher and in microsuspension assay 45-fold higher with respect to strain TA98. In the presence of S9 mix, the mutagenic potency in YG1041 declined (P<0.001) in summer, but increased in TA98 (P<0.05) in samples collected during the winter season. The microsuspension assay provided higher mutagenic responses in both tester strains, but in both strains a significant decrease of mutagenic potency was observed in the presence of S9 mix (P<0.001 for YG1041, P<0.05 for TA98 in winter). The mutagenic potencies detected with both indicator strains correlated well (r=0.54 to 0.87) within each mutagenicity test used but not (for TA98) or moderately (r=0.44 to 0. 66 for YG1041) between both of the tests. The mutagenic activity (in rev/m(3)) likewise the concentration of benzo[a]pyrene and sum of carcinogenic PAHs showed seasonal variation with distinctly higher values during winter season. A correlation between the PAH concentrations and the mutagenicity results for the plate incorporation, but not for the microsuspension tests was found. In samples from higher industrial areas, the higher mutagenicity values were obtained in plate incorporation test with TA98 and in both tests with YG1041 in summer season (P<0.05). According to our results, plate incorporation test seems to be more informative than microsuspension assay. For routine ambient air mutagenicity monitoring, the use of YG1041 tester strain without metabolic activation and the plate incorporation test are to be recommended.     

Comprehensive assessment of the photomutagenicity, photogenotoxicity and photo(cyto)toxicity of azulene

The polycyclic aromatic hydrocarbon azulene and its naturally occurring derivative guaiazulene (1,4-dimethyl-7-isopropylazulene) are known to absorb light in the UV-vis region of the spectrum. Both compounds were reported to be mutagenic in the Salmonella typhimurium bacterial mutagenicity assay (Ames test) in strain TA102, and to cause DNA damage in the comet assay in vitro upon exposure to UVA light. In contrast, another study reported a photoprotective effect in vitro of guaiazulene. We present here a comprehensive assessment of the photo(cyto)toxicity (3T3 fibroblast Neutral Red uptake test), the photomutagenicity (Ames test) and photogenotoxicity (comet assay and micronucleus test in L5178Y cells in vitro) of azulene. In the Ames test, the mutagenicity of azulene was assessed in the presence and absence of UV light by use of the Salmonella strains TA102, TA104, TA2638 and E. coli WP2. Azulene was irradiated before being plated with bacteria (pre-irradiation), or concomitantly with the bacteria either after plating or while in suspension. Guaiazulene was included in some of the experiments. Neither in the photo-Ames test nor in the other photogenotoxicity tests, azulene or guaiazulene showed any photomutagenic or photogenotoxic activity. Weak photo(cyto)toxicity (estimate of PIF≥1.67) was observed with azulene in the 3T3 NRU test, the Alamar Blue test and the relative cell count, which may be due to the generation of reactive oxygen species, as reported recently.     

Successive Mineralization and Detoxification of Benzo[a]pyrene by the White Rot Fungus Bjerkandera sp. Strain BOS55 and Indigenous Microflora

White rot fungi can oxidize high-molecular-weight polycyclic aromatic hydrocarbons (PAH) rapidly to polar metabolites, but only limited mineralization takes place. The objectives of this study were to determine if the polar metabolites can be readily mineralized by indigenous microflora from several inoculum sources, such as activated sludge, forest soils, and PAH-adapted sediment sludge, and to determine if such metabolites have decreased mutagenicity compared to the mutagenicity of the parent PAH. ¹⁴C-radiolabeled benzo[a]pyrene was subjected to oxidation by the white rot fungus Bjerkandera sp. strain BOS55. After 15 days, up to 8.5% of the [¹⁴C]benzo[a]pyrene was recovered as ¹⁴CO₂ in fungal cultures, up to 73% was recovered as water-soluble metabolites, and only 4% remained soluble in dibutyl ether. Thin-layer chromatography analysis revealed that many polar fluorescent metabolites accumulated. Addition of indigenous microflora to fungal cultures with oxidized benzo[a]pyrene on day 15 resulted in an initially rapid increase in the level of ¹⁴CO₂ recovery to a maximal value of 34% by the end of the experiments (>150 days), and the level of water-soluble label decreased to 16% of the initial level. In fungal cultures not inoculated with microflora, the level of ¹⁴CO₂ recovery increased to 13.5%, while the level of recovery of water-soluble metabolites remained as high as 61%. No large differences in ¹⁴CO₂ production were observed with several inocula, showing that some polar metabolites of fungal benzo[a]pyrene oxidation were readily degraded by indigenous microorganisms, while other metabolites were not. Of the inocula tested, only PAH-adapted sediment sludge was capable of directly mineralizing intact benzo[a]pyrene, albeit at a lower rate and to a lesser extent than the mineralization observed after combined treatment with white rot fungi and indigenous microflora. Fungal oxidation of benzo[a]pyrene resulted in rapid and almost complete elimination of its high mutagenic potential, as observed in the Salmonella typhimurium revertant test performed with strains TA100 and TA98. Moreover, no direct mutagenic metabolite could be detected during fungal oxidation. The remaining weak mutagenic activity of fungal cultures containing benzo[a]pyrene metabolites towards strain TA98 was further decreased by subsequent incubations with indigenous microflora.     

High mutagenic potency of several polycyclic aromatic hydrocarbons induced by liver postmitochondrial fractions from control and xenobiotic-treated immature carp

The metabolism of carcinogens in fish was examined by measuring the activation of different polycyclic aromatic hydrocarbons (PAH) by carp (Cyprinus carpio L.) liver post-mitochondrial fractions (S9) using the Salmonella typhimurium TA100 reverse mutation assay. For this study, 1 non-carcinogen, anthracene (AN), and 4 carcinogens, chrysene (CHR), benzo[a]pyrene (BaP), 3-methylcholanthrene (3MC) and 7,12-dimethylbenzanthracene (DMBA), were chosen. The bioactivating potency of the metabolic systems of carp pretreated with phenobarbital (PB), 3MC or Aroclor 1254 (ARO) were compared to uninduced carp liver. The results show that carp liver has the ability to metabolize carcinogenic PAH into mutagenic metabolites, which is enhanced when carp are pretreated with 3MC or ARO, but not with PB. A positive correlation between the induction of aryl hydrocarbon hydroxylase (AHH) activity in carp liver and the mutagenic potencies of CHR, BaP, DMBA and 3MC, has been observed. The bioactivating ability of carp liver S9 was compared with the ability of the same fractions from female Wistar rats (this study) as well as from Sprague-Dawley rats (literature data). When the mutagenic potencies of selected PAH had been normalized on the activity of BaP, the following order of mutagenic activities with S9 fractions from ARO-treated animals was obtained: (1) BaP (1) greater than DMBA (0.26) greater than 3MC (0.22) greater than CHR (0.05) greater than AN (0) for carp; (2) BaP (1) greater than 3MC (0.48) greater than CHR (0.31) greater than DMBA (0.16) greater than AN (0) for Sprague-Dawley rats; and (3) BaP (1) greater than 3MC (0.17) greater than DMBA (0.11) greater than CHR (0) = AN (0) for female Wistar rats. We conclude that carp and rats are very similar in their ability to activate carcinogenic PAH into mutagenic metabolites, which suggests that carp may be very susceptible to the carcinogenic activity of these compounds. According to our results from the mutagenicity study, as well as from the enzyme induction study, we propose the use of carp as a suitable model system for the study of chemical carcinogens.