Conjugation of benzo[a]pyrene metabolites by freshwater green alga Selenastrum capricornutum

Benzo[a]pyrene (BaP) undergoes metabolic transformation in mammals via oxidative, hydrolytic, and conjugative processes; however, little is known concerning BaP conjugation in freshwater algae. It has been shown in this laboratory that BaP is metabolized by Selenastrum capricornutum via a dioxygenase pathway. This study describes the conjugation of BaP metabolites by a green alga, Selenastrum capricornutum. Cultures were exposed to 1160 micrograms/l [14C]BaP for 4 days at 23 degrees C under gold fluorescent lights on a diurnal cycle of 16 h light, 8 h dark. Of the total metabolites in the algal culture, 89% were present in media. BaP and non-conjugated metabolites were separated from conjugated metabolites by chromatography on neutral alumina columns using solvents of increasing polarity. Seventy-one percent of the BaP metabolites were conjugates of which 12.2%, 12.0% and 12.4% were sulfate ester and alpha- and beta-glucose conjugates, respectively. Conjugates that coeluted with sulfate esters were hydrolyzed with arylsulfatase, alpha- or beta-glucosidase; high performance liquid chromatography (HPLC) analysis indicated that the major product of each enzymatic hydrolysis was the 4,5-dihydrodiol (87.2, 69 and 53%, respectively). Eighty-six percent of the conjugates were acid labile following incubation for 2 h in 4 N HCl at 37 degrees C. To our knowledge this is the first demonstration of the metabolism of a polynuclear aromatic hydrocarbon by a freshwater green alga through a dioxygenase pathway and subsequent conjugation and excretion.     

Removal and transformation of high molecular weight polycyclic aromatic hydrocarbons in water by live and dead microalgae

The removal and transformation of seven high molecular weight polycyclic aromatic hydrocarbons (PAHs), namely benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, indeno[1,2,3-c,d]pyrene and benzo[g,h,i]perylene, by a freshwater microalga Selenastrum capricornutum under gold and white light irradiation was studied. The two light sources did not result in significant differences in the biodegradation of the selected PAHs in live algal cells, but white light was more effective in promoting photodegradation than was gold light in dead cells. The removal efficiency of seven PAHs, as well as the difference between live and dead microalgal cells, was PAH compound-dependent. Benz[a]anthracene and benzo[a]pyrene were highly transformed in live and dead algal cells, and dead cells displayed greater transformation levels than live cells. Further investigation comparing the transformation of single PAH compound, benzo[a]pyrene, by S. capricornutum and another green microalgal species, Chlorella sp., demonstrated that the transformation in dead cells was similar, indicating the process was algal-species independent. Dead algal cells most likely acted as a photosensitizer and accelerated the photodegradation of PAHs.     

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.     

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.     

Influence of the carcinogenic pollutant benzo[a]pyrene on plant development: fern gametophytes

Polycyclic aromatic hydrocarbons (PAHs), ubiquitous environmental pollutants, are known to be biologically active in mammalian systems and are accumulated by plants. A few reports suggest that PAHs stimulate growth in plants and induce morphogenesis in plant tissue in culture. To investigate the growth altering abilities of PAHs in plants, polypodiaceous fern gametophytes were grown under sterile conditions on media containing a biologically active PAH, benzo[a]pyrene (BaP), at doses ranging from 0.1 to 10.0 micrograms/ml. The growth pattern of polypodiaceous fern gametophytes enables alterations in growth and morphogenesis to be observed at the cellular level in an intact plant. Doses of BaP in the range 0.1--3.2 micrograms/ml enhanced the onset of the morphological transitions from 1-dimensional (1D) to 2-dimensional (2D) growth. This transition for BaP treated plants occurred after fewer cell divisions than the corresponding solvent and untreated controls. The low (0.1 and 0.32 micrograms) and high (1.0 and 3.2 micrograms) doses of BaP were found to accelerate and inhibit cell proliferation, respectively. The 10.0 microgram dose was toxic and resulted in decreased germination of spores and reduced survival of plants. A slight but significant decrease in survival was also observed in ferns treated with 3.2 micrograms. This is the first example of PAH influencing cell differentiation in a whole plant system.     

Sensitivity of different bacterial assays in detecting mutagens in urine of humans exposed to polycyclic aromatic hydrocarbons.

The urine mutagenicity and excretion of 1-hydroxypyrene (1-OH PYR) in non-smoking psoriatic patients treated topically with coal-tar-based ointments were analysed in order to find the most appropriate procedure for monitoring occupational PAH exposure. The bacterial mutagenicity assays used were the plate incorporation, macro-scale fluctuation and microsuspension tests, all on Salmonella typhimurium strain TA98 in the presence of S9 mix and beta-glucuronidase. The sensitivities of the three assays in detecting mutagenic urinary PAH metabolites were compared. The efficiencies of XAD-2 and C18 resins for concentrating PAH urinary mutagens were evaluated in the microsuspension assay. The plate and fluctuation tests on XAD-2 urine extracts were shown to be insufficiently sensitive to detect low urinary levels of mutagens, being positive on urine samples with very high PAH metabolite content, estimated as more than 30 micrograms/g of creatinine of 1-OH PYR. The microsuspension assay on XAD-2 or, even better, on C18 urine extracts was very sensitive in detecting up to 5 micrograms/g of creatinine of 1-OH PYR. It therefore seems to be applicable to the biological monitoring of most occupational low exposures to coal tar.     

Mutagenicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and benzo[a]pyrene (BaP) in the gill and hepatopancreas of rpsL transgenic zebrafish

We examined the in vivo mutagenicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and benzo[a]pyrene (BaP) by using transgenic (Tg) zebrafish carrying the mutational target gene rpsL. PBTA-6 is one of the PBTA-type compounds that were recently identified in highly mutagenic river water in Japan. BaP is a well-known contaminant that is frequently found in polluted water. Both compounds are potent mutagens, as determined by using the Ames test employing S9 mix and Salmonella. Adult rpsL Tg zebrafish were exposed to 0, 7, or 10 mg/L PBTA-6 or 0, 1.5, or 3 mg/L BaP for 96 h in a water bath and the mutations in their gills and hepatopancreata were measured 2-4 weeks later. At 3 weeks after exposure, 3 mg/L BaP significantly increased the rpsL mutant frequency (MF) in the gill and hepatopancreas by 5- and 2.3-fold, respectively, as compared to control fish. Sequence analysis showed that BaP mainly induced G:C to T:A and G:C to C:G transversions, which is consistent with the known mutagenic effects of BaP. In contrast, despite its extremely high mutagenic potency in Salmonella strains, PBTA-6 did not significantly increase the MF in the zebrafish gill or hepatopancreas. Although PBTA-6 is 300 times more mutagenic than BaP in the Ames test [T. Watanabe, H. Nukaya, Y. Terao, Y. Takahashi, A. Tada, T. Takamura, H. Sawanishi, T. Ohe, T. Hirayama, T. Sugimura, K. Wakabayashi, Synthesis of 2-phenylbenzotriazole-type mutagens, PBTA-5 and PBTA-6, and their detection in river water from Japan, Mutat. Res. 498 (2001) 107-115], calculation of the mutagenicity per mole of compound indicated that PBTA-6 was 33- and <3.7-fold less mutagenic in the zebrafish gill and hepatopancreas, respectively, than BaP.     

Fungal metabolism and detoxification of polycyclic aromatic hydrocarbons

The mutagenic activity of ethyl acetate extracts of culture medium from Cunninghamella elegans incubated 72 h with various polycyclic aromatic hydrocarbons (PAHs) was evaluated in the Salmonella typhimurium reversion assay. All of the PAH extracts were assayed in tester strains TA98 and TA100 both with and without metabolic activation using a liver fraction from Aroclor 1254-treated rats. None of the extracts from fungal incubations with the mutagenic PAHs, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene and benz[a]anthracene, as well as the non-mutagenic PAHs, naphthalene, phenanthrene and anthracene, displayed any appreciable mutagenic activity. In addition, time course experiments indicated that the rate of decrease in mutagenic activity in the extracts from cultures incubated with benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene was coincident with the rate of increase in total metabolism. The results demonstrated the ability of the fungus C. elegans to detoxify known carcinogens and mutagens and suggests that this organism may play an important role in the metabolism and inactivation of PAHs in the environment.Abbreviations hplc high performance liquid chromatography - tlc thin layer chromatography - PAH polycyclic aromatic hydrocarbon     

Mutagenicity of food pellets from human diets in The Netherlands

Food pellets from human diets, prepared according to mean consumption figures in The Netherlands, were assessed on mutagenicity and mutagens were identified. Three types of human meals were compared: raw (C), heated (D) and heated with vegetables and fruit (E, a complete meal). In addition 2 animal diets were tested: commercial control diet (A), and a control diet to which vegetables and fruit had been added (B). All human diets contained: 40.6 energy (E)% fat, 13.2 E% protein, 46.2 E% carbohydrate and 5.2% (w/w) fibre. For animal diets these figures were 21.6, 26.0, 52.4 and 10.7% respectively. After extraction samples were tested in the Salmonella-microsome test, tester strains TA1538, TA98 and TA100. Human diets with heated products (D, E) were both clearly mutagenic with approximately 300-500 revertants per gram. Food pellets from animal diets (A, B) displayed no mutagenic activity. HPLC-derived chromatographic fractions of diets D and E showed 3 large mutagenic areas identified as IQ (2-amino-3 methyl-imidazo-[4,5-f]quinoline) and MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline and PhIP (2-amino-6-phenylimidazo[4,5-b]pyridine) and other mutagens not completely defined. This mutagen profile was similar to that found previously for fried beef. Mass estimates for these potent mutagens amounted to 15-20 micrograms/kg. Health implications of these findings are discussed. As IQ, MeIOx and DiMeIQx have been found to be weakly carcinogenic in rodents and many other initiating and modulating factors may be present in a complex human diet, a chronic toxicity study is indicated.     

Experimental Studies on the Physiology of the Phycobiont and Mycobiont Ramalina ecklonii By

The lichenized fungus and alga of the fruticose lichen Ramalini ecklonii were isolated into pure cultures. The ascospores of the fungus failed to germinate in less than five weeks incubation in spite of the use of a variety of cultural conditions. The fungus showed a considerable increase in growth on malt extract agar. Both organisms showed a marked tolerance for high concentrations of glucose although growth was quantitatively reduced. The fungus was able to use a variety of carbon and nitrogen sources as well as an extract of algal cells. Cultivation in the absence of biotin and thiamine failed to yield significant amounts of growth. The alga yielded 27 mg of dry weight after three weeks in a synthetic medium under low light intensities. The alga could be grown in satisfactory amounts on CO₂ and inorganic salts with moderate light intensities. Experiments using ¹⁴CO₂ showed the fungus able to incorporate the extra-cellular and intra-cellular products of algal metabolism. The rate of incorporation of extra-cellular products was inhibited by high concentrations of biotin and thiamine. The alga assimilated ^l4CO₂ which was retained by the cells over a period of 14 days, at which time 78 per cent of the activity was insoluble in 80 per cent ethanol. An extract of the fungus labelled with ¹⁴C glucose was partially taken up by the alga and 50 per cent of the label was insoluble in 80 per cent after three days incubation in the light. No lichen acids were found in either the fungal cultures or the algal cultures although large amounts (e.g. 2 liters) of material were extracted and chromatographed. Usnic acid was produced by the intact lichen thallus.     

Evaluating the relationship of metabolic activation system concentrations and chemical dose concentrations for the Salmonella spiral and plate assays

A factorial experimental design was used within this study to evaluate the influence of multiple metabolic activation system concentrations on the dose-response exhibited by promutagens (indirect-acting mutagens) in the Salmonella spiral and plate assays. The mutagenic activity of the three compounds used spanned three orders of magnitude. The mutagenic activity of the compounds ranged from 10 to 100 revertants/micrograms for acetylaminofluorene (2AAF) to more than 1000 revertants/micrograms for 2-aminoanthracene (2AA). Benzo [a] pyrene (BaP) activity was within an intermediate range (100-1000 revertants/micrograms). During a single experiment, a mutagen was tested in TA100 at 13 doses plus a negative control dose. Each dose was tested at 10 S9 concentrations. The S9 concentrations ranged from 0.1 mg protein/plate to 4 mg protein/plate in the standard plate assay and from 0.25 to 4.90 mg-equivalents in the spiral assay. The spiral Salmonella assay, an automated version of the standard assay, generates dose-response data from a concentration gradient on a single agar plate, thereby providing a straightforward approach to this type of study. This study demonstrates not only that even small differences in S9 concentrations can affect the measurement of mutagenic potency but that S9/compound interactions cannot be generalized through the use of interaction studies. This study also shows that spiral assay data and plate assay data for promutagens cannot be compared directly unless the S9 concentrations for all chemical doses are also comparable.     

Effects of Oil Refinery Effluents on Selenastrum capricornutum PRINTZ

Laboratory-based bioassay experiments using Selenastrum capricornutum PRINTZ as the test organism were conducted to evaluate the potentiality of refinery effluents to sustain algal growth. The raw effluents were remarkably toxic to the test alga, but satisfactory algal growth occurred in (diluted) 0.01 % effluents. The effluents from subsequent treatment stages required much less dilution to eliminate the toxicity. This study implicates oil and phenol in the reduction of algal growth in refinery effluents. A highly significant negative correlation was found between the final yield of the test alga and the concentration of oil or phenol in the culture suspension. The bioassay experiments therefore point to the toxic nature of refinery effluents. On the basis of these findings it is recommended that proper dilution of refinery effluents is necessary before they are discharged into any body of water.     

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.     

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.     

Photomophogenesis in the Blue-green Alga Nostoc commune 584

The blue-green alga Nostoc commune 584 displays a photocontrolled developmental cycle similar to that described for N. muscorum A by Lazaroff and Vishniac (1961). In both species white fluorescent light acts at the same stage, ragulating the development of motile trichomes from sheathed aseriate colonies. However white light blocks this step in N. commune 584, whereas the formation of motile trichomes is promoted by white light in N. muscorum A. Light-grown (aseriate) cultures in N. commune 584 were used to determine the action spectra for photomorphogenesis. Green light (max 520 nm) inhbited aseriate colony breakage, and red light (max 640 nm) promoted colony breakage and the differentiation of motile trichomes. On a quantum basis green light was about 3 times more effective than red light. The morphogenetic effects of either red or green light were reversible by irradiation with the other color of light. Repeated photoreversibility was observed, and the algal culutres responded only to the color of the last irradiation in a sequence. An unidentified substance is excreted into the media of motile cultures of both N. commune 584 and N. muscorum A which promotes motility in non-motile cultures. The motility-promoting substances from both species are reciprocally active. Activity is lost when the media are autoclaved.     

Lipid peroxidation and benzo[a]pyrene activation to mutagenic metabolites: in vivo influence of vitamins A, E and C and glutathione in both dietary vitamin A sufficiency and deficiency

Rats fed with either a sufficient-vitamin A or a vitamin A-free diet were pretreated with 750 mg/kg body weight of retinyl palmitate, alpha-tocopherol acetate, ascorbic acid or glutathione. Benzo[a]pyrene (BaP) metabolism and BaP-induced mutagenesis in Salmonella typhimurium TA98 were investigated and related to lipid peroxidation activities in postmitochondrial (S9) liver fraction. The microsomal mixed-function oxidase activities were decreased by vitamin A deficiency and weakly affected by scavenger treatment. The rate of lipid peroxidation of microsomal membranes was unaffected by vitamin A deficiency because of decreased polyunsaturated fatty acids and increased vitamin E contents. However, lipid peroxidation was decreased by pretreatment with fat-soluble vitamins (chiefly vitamin E) and increased by ascorbic acid. Within each experimental group both BaP metabolism and BaP mutagenic activity were closely correlated with the rate of lipid peroxidation. In vitamin A deficiency, the increased BaP metabolism and mutagenicity could be related to a decrease in cytosolic contents of scavengers (vitamin A and glutathione). In Ames test conditions, the free radical pathway became a route for BaP metabolism and thus the BaP activation to mutagenic metabolites is related to the cellular status in free radical scavengers.     

A modified Vibrio harveyi mutagenicity assay based on bioluminescence induction

AIMS: Mutagenic pollution of natural environment is currently one of the most serious ecological problems. Therefore, rapid detection of the presence of mutagens is a very important issue. Although many mutagenicity assays have already been described, only a few are suitable for testing samples from natural environment. One of such assays is a microbiological mutagenicity test based on genetically modified Vibrio harveyi strains. The aim of this work was to modify and improve the V. harveyi assay. METHODS AND RESULTS: A series of V. harveyi dark and dim mutants were tested for reversion of their phenotype towards efficient light emission in response to incubation with known mutagens. Luminescence of the A16 strain (luxE mutant) increased significantly after a few hours of such a treatment with various mutagenic agents, revealing a dose-response correlation. Sensitivity of the assay has been determined for different mutagens. CONCLUSIONS: The luminescence-based V. harveyi mutagenicity assay is rapid, sensitive and reveals a dose-response correlation for various mutagens. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed in this study is a potentially useful tool in studies on mutagenic pollution of environment, especially marine water.     

A comparative study on the metabolic activation of 3,4-benzo[a]pyrene to mutagens by aorta smooth muscle cells of rat and rabbit

The mutagenic activation of benzo[a]pyrene (BaP) after exposure to aorta smooth muscle cells of different origin was examined. Three test systems with different genetic endpoints--sister-chromatid exchange (SCE), gene mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus and unscheduled DNA synthesis (UDS)--were used. Treatment of rat and rabbit aorta smooth muscle cells with BaP (1-6 micrograms/ml) resulted in a significant increase of SCEs, HGPRT mutations and UDS. So smooth muscle cells are capable of converting BaP to metabolites with a DNA-damaging action. In order to investigate the relation between the formation of mutagenic BaP metabolites and the susceptibility to atherosclerosis we compared the mutagenic potential of BaP using aorta smooth muscle cells of different species (rat, rabbit) and locations (thoracic and abdominal aorta). Rabbits and abdominal aortas are more susceptible to atherosclerosis than rats and thoracic aortas. The SCE, HGPRT and UDS assays revealed that smooth muscle cells of different origin possessed the same metabolic potential towards BaP. There was no correlation between the mutagenic potency of BaP metabolites and the susceptibility to atherosclerosis. As smooth muscle cells have a low metabolic capacity towards BaP, probably other factors in addition to the metabolic capacity of smooth muscle cells are responsible for species and tissue differences in susceptibility to atherosclerosis.     

The use of algal assays and chlorophyll concentrations to determine fertility of water in small impoundments in West Virginia

Seven small artificial lakes in West Virginia were sampled in the spring and fall. Water taken during these isothermal periods was used for the EPA Algal Assay with Selenastrum capricornutum as the test alga. In all cases the limiting nutrient was phosphorus. However in every case the water was fertile enough for the alga to grow well without the addition of nutrients. Each lake was sampled several times during the summer and nutrient analysis and chlorophyll concentrations indicated that each lake is probably eutrophic. These conclusions were borne out by the algal assays. The assays also indicated that there were probably no toxic elements in any of the seven lakes.