Terence E. Cody

Biotransformation of benzo[a]pyrene and other polycyclic aromatic hydrocarbons and heterocyclic analogs by several green algae and other algal species under gold and white light.

This laboratory has shown that the metabolism of benzo[a]pyrene (BaP), a carcinogenic polycyclic aromatic hydrocarbon (PAH), by a freshwater green alga, Selenastrum capricornutum, under gold light proceeds through a dioxygenase pathway with subsequent conjugation and excretion. This study was undertaken to determine: (1) the effects of different light sources on the enzymatic or photochemical processes involved in the biotransformation of BaP over a dose range of 5-1200 mg/l; (2) the phototoxicity of carcinogenic PAHs and mutagenic quinones to a green alga; (3) the ability of other algal systems to metabolize BaP. Cultures were exposed to different doses of BaP for 2 days at 23 degrees C under gold, white or UV-A fluorescent light on a diurnal cycle of 16 h light, 8 h dark. Under gold light, metabolites of BaP produced by Selenastrum capricornutum were the dihydrodiols of which the 11,12-dihydrodiol was the major metabolite. Under white light, at low doses, the major metabolite was the 9,10-dihydrodiol. With increasing dose, the ratio of dihydrodiols to quinones decreased to less than two. With increasing light energy output, from gold to white to UV-A in the PAH absorbing region, BaP quinone production increased. Of other carcinogenic PAHs studied, only 7H-dibenz[c,g]carbazole was as phototoxic as BaP while 7,12-dimethylbenz[a]anthracene, dibenz[a,j]acridine and non-carcinogenic PAHs, anthracene and pyrene, were not phototoxic. The 3,6-quinone of BaP was found to be highly phototoxic while quinones that included menadione, danthron, phenanthrene-quinone and hydroquinone were not. The data suggest that the phototoxicity of BaP is due to photochemical production of quinones; the 3,6-quinone of BaP is phototoxic and is probably the result of the production of short lived cyclic reactive intermediates by the interaction of light with the quinone. Lastly, only the green algae, Selenastrum capricornutum, Scenedesmus acutus and Ankistrodesmus braunii almost completely metabolized BaP to dihydrodiols. The green alga Chlamydomonas reinhardtii, the yellow alga Ochromonas malhamensis, the blue green algae Anabaena flosaquae and euglenoid Euglena gracilis did not metabolize BaP to any extent. The data indicate that algae are important in their ability to degrade PAHs but the degradation is dependent on the dose of light energy emitted and absorbed, the dose of PAHs to which the algae are exposed, the phototoxicity of PAHs and their metabolite(s) and the species and strain of algae involved. All of these factors will be important in assessing the degradation and detoxification pathways of recalcitrant PAHs by algae.

Metabolism of mutagenic polycyclic aromatic hydrocarbons by photosynthetic algal species

Polycyclic aromatic hydrocarbons (PAH) known to produce carcinogenic and mutagenic effects have been shown to contaminate waters, sediments and soils. While it is accepted that metabolites of these compounds are responsible for most of their biological effects in mammals, their metabolism, and to a large extent their bioactivity, in aquatic plants have not been explored. Cultures of photosynthetic algal species were assayed for their ability to metabolize benzo[a]pyrene (BaP), a carcinogenic PAH under conditions which either permitted (white light) or disallowed (gold light) photooxidation of the compound. Growth of Selenastrum capricornutum, a fresh-water green alga, was completely inhibited when incubated in white light with 160 micrograms BaP/l medium. By contrast concentrations at the upper limit of BaP solubility in aqueous medium had no effect on algal growth when gold light was used. BaP quinones and phenol derivatives were found to inhibit growth of Selenastrum under white light incubation. BaP phototoxicity and metabolism were observed to be species-specific. All 3 tested species of the order Chlorococcales were growth-inhibited by BaP in white light whereas neither the green alga Chlamydomonas reinhardtii nor a blue-green, a yellow-green or an euglenoid alga responded in this fashion. Assays of radiolabeled BaP metabolism in Selenastrum showed that the majority of radioactivity associated with BaP was found in media as opposed to algal cell pellets, that the extent of metabolism was BaP concentration dependent, and that the proportion of various metabolites detected was a function of the light source. After gold light incubation, BaP diols predominated while after white light treatment at equal BaP concentrations, the 3,6-quinone was found in the highest concentration. Extracted material from algal cell pellets and from media was tested for mutagenicity in a forward mutation suspension assay in Salmonella typhimurium using resistance to 8-azaguanine for selection. Direct-acting mutagens were detected in extracted media from incubation of Selenastrum with 400 micrograms BaP/l for 1 day in gold light. Extracts of media from algae incubated in gold light from 1 to 4 days with 1200 micrograms BaP/l were found to have direct-acting mutagens as well as those requiring further metabolism. Media extracts from white light incubations of BaP were mutagenic upon addition of rat liver homogenates. Activity of these materials from white light treatment are largely attributable to unmetabolized BaP.

The phototoxicity of benzo[a]pyrene in the green alga Selenastrum capricornutum☆

The effects of selected polycyclic aromatic hydrocarbons (PAHs) on the growth of the green alga Selenastrum capricornutum in three light regimens were examined. In gold fluorescent light, benzo[a]pyrene (BaP) at 12 mg/liter (48 mumole/liter), benz[a]anthracene (BaA) at 40 mg/liter (175 mumole/liter), anthracene (A) at 40 mg/liter (224 mumole/liter), and 13 metabolites of BaP each at 40 micrograms/liter had no effect on algal growth. In cool-white fluorescent light, 30% inhibition of algal growth occurred with 0.1 mumole/liter BaP, 8.0 mumole/liter BaA, and 40 mumole/liter A. BaP at 0.16 mg/liter (0.64 mumole/liter) totally inhibited growth. BaP concentrations an order of magnitude lower inhibited algal growth in fluorescent blacklight. In cool-white light, 5 of 13 metabolites of BaP (each 40 micrograms/liter) inhibited algal growth: 3,6-quinone; 6-hydroxy; 9-hydroxy; 3-hydroxy; and 1,6-quinone. Based on these results, PAHs and metabolites of BaP are selectively phototoxic to S. capricornutum due to the incident light intensity below 550 nm.

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.

Isolation, characterization, and long-term culture of fetal bovine tracheal epithelial cells

SummaryEpithelial cells were isolated from fetal bovine trachea by exposing and stripping the mucosal epithelium from the adjacent connective tissue. The tissue was minced and enzymically dissociated in Ca-Mg-free medium containing dispase and dithiothreitol. The stripping procedure and selective trypsinization produced epithelial cell cultures free of fibroblasts. Seeded on plastic, the plating efficiency was 21.5% with a doubling time of 24 h. Dome formation, evidence of occluding junctions and active ion transport characteristic of epithelial cells, was common. Growth of the cells on glass, collagen, and Engelbreth-Holm-Swarm (EHS) substrate demonstrated a striking difference in morphology. Cells grown on EHS presented a more distinctly three-dimensional growth pattern and many more microvilli when compared to cells grown on glass or collagen. The cells retained their epithelioid characteristics through more than 30 passages as shown by the presence of distinct apical and basolateral membranes, tight junctions, and positive keratin staining.

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.

Cell proliferation and nuclear abnormalities are increased and apoptosis is decreased in the epidermis of the p53 null mouse after topical application of benzo[a]pyrene

Abstract. Cell proliferation and cell death in mouse epidermis are altered by topical application of benzo[a]pyrene (BaP), a procarcinogen, which yields metabolites that can form DNA adducts. The mitotic rate, nuclear abnormalities, labelling index, grain density, necrosis and apoptosis were compared in the epidermis of TSG‐p53 null (p53‐/‐) and C57BL wild‐type (wt) mice after weekly treatments with BaP to determine whether the absence of the p53 gene altered cytokinetic responses to DNA damaging agents in vivo. Acetone alone or 64 μg BaP in 50 μl acetone was applied to the clipped dorsum of mice once, or in four consecutive weekly treatments. Indices of cell proliferation and cell death were the same in both wt and p53‐/‐ mice treated only with acetone. One application of BaP depressed mitosis and slowed the rate of DNA synthesis in both genotypes. After four applications of BaP the number of keratinocytes in S phase increased substantially, while there was no further slowing in the rate of S phase in the wt and p53‐/‐ mice. Cell proliferation rates and numbers of cells with nuclear abnormalities were higher and there were fewer apoptotic cells and apoptotic bodies in the p53‐/‐ mice than in the wt mice. Numbers of ‘sunburn’cells were similar in both types.

Colcemid alters s phase and other parameters in skin during chronic exposure to benzo(a)pyrene

The administration of Colcemid for collecting mitotic figures in a carcinogenesis study, using benzo(a)pyrene (BaP), diminished the experimental differences between exposed and control mice. A dose‐related increase in noncollected mitotic index (n‐mitotic index) was seen in keratinocytes in the dorsal epidermis of mice which received four weekly treatments of BaP at 16, 32 and 64 μg in 50 μl of acetone. In contrast, the number of mitotic figures collected for 4 hr by Colcemid block (c‐mitotic index) was depressed at 16 μg, unchanged at 32 μg, and elevated at 64 μg of BaP. Weekly treatments with 4, 8 or 16 μg BaP for 3–8 months induced an elevation in both n‐mitotic and c‐mitotic indices. The differences in results produced by the two methods of determining mitotic index depended upon dose and duration of treatment with BaP. The administration of Colcemid to acetone‐treated mice increased the labeling index (number of labeled cells) and reduced the rate of DNA synthesis (low grain count per keratinocyte nucleus). After chronic application of BaP, Colcemid abrogated the increase in labeling index, but produced no additional effect on the number of grains per labeled keratinocyte. The modifying effect of Colcemid was greatest when administered during the peak of the tissue response to BaP. A number of significant changes in morphology of the skin associated with chronic exposure to BaP were attenuated by the use of Colcemid.