David Warshawsky

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.

Biodegradation of carbazole by Ralstonia sp. RJGII.123 isolated from a hydrocarbon contaminated soil

The use of microorganisms for bioremediation of contaminated soils may be enhanced with an understanding of the pathways involved in their degradation of hazardous compounds. Ralstonia sp. strain RJGII.123 was isolated from soil located at a former coal gasification plant, based on its ability to mineralize carbazole, a three-ring N-heterocyclic pollutant. Experiments were carried out with strain RJGHII.123 and 14C-carbazole (2 mg/L and 500 mg/L) as the sole organic carbon source. At 15 days, 80% of the 2 mg/L carbazole was recovered as CO2, and <1% remained as undegraded carbazole, while 24% of the 500 mg/L carbazole was recovered as CO2 and approximately 70% remained as undegraded carbazole. Several stable intermediates were formed during this time. These intermediates were separated by high performance liquid chromatography (HPLC) and were characterized using high resolution mass spectroscopy (HR-MS) and gas chromatography - mass spectroscopy (GC-MS). At least 10 ring cleavage products of carbazole degradation were identified; four of these were confirmed as anthranilic acid, indole-2-carboxylic acid, indole-3-carboxylic acid, and (1H)-4-quinolinone by comparison with standards. These data indicate that strain RJGII.123 shares aspects of carbazole degradation with previously described Pseudomonas spp., and may be useful in facilitating the bioremediation of NHA from contaminated soils.

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.

Comparative Carcinogenicity, Metabolism, Mutagenicity, and DNA Binding of 7H-Dibenzo[c,g]carbazole and Dibenz[a,j]acridine

Complex mixtures that are produced from the combustion of organic materials have been associated with increased cancer mortality. These mixtures contain homocyclic and heterocyclic polycyclic aromatic hydrocarbons (PAHs), many of which are known carcinogens. In particular, N-heterocyclic aromatic compounds (NHA) are present in these mixtures. Studies to determine the metabolic activation of these compounds have been undertaken. The purpose of this review is to compare and contrast the metabolic activation and biological effects of two NHA, 7H-dibenzo[c,g]carbazole (DBC) and dibenz[a,j]acridine (DBA), in order to better assess the contribution of NHA to the carcinogenic potency of complex mixtures and to develop biomarkers of the carcinogenic process. DBC has both local and systemic effects in the mouse; it is a potent skin and liver carcinogen following topical application and a lung carcinogen following i.p. application. On the other hand, DBA is a moderate mouse skin carcinogen following topical application and a lung carcinogen following subcutaneous injection. The biological differences for DBC and DBA are reflected in target organ-specific proximate and mutagenic metabolites and DNA adduct patterns.

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.

Comparative metabolism of 7H-dibenzo[c,g]carbazole and dibenz[a,j]acridine by mouse and rat liver microsomes

The comparative metabolism of the carcinogenic pollutants 7H-dibenzo[c,g]-carbazole (DBC) and dibenz[a,j]acridine (DBA) was investigated in vitro using 3-methylcholanthrene (3MC) induced Sprague-Dawley rat and Hsd:ICR(Br) mouse liver microsomal preparations with benzo[a]pyrene (BaP) as the positive control. Metabolites were isolated and separated by HPLC and identified by spectroscopic and co-chromatographic techniques using synthetic standards. The major metabolites of DBC were the phenols: the 5-OH-DBC, 3-OH-DBC, and 2-OH-DBC. Traces of 1-OH-DBC were also found yet no dihydrodiols were identified. The major metabolites of DBA were the 3,4-diol-DBA and 5,6-diol-DBA, 1,2-diol-DBA, DBA-5,6-oxide and 4-OH-DBA. Treatment of both mice and rats with 3MC resulted in significant (P less than or equal to 0.05) increases relative to control in the microsomal metabolism of DBA to dihydrodiol and phenol metabolites, similar to that observed for BaP. 3MC-induced rat liver microsomes significantly (P less than or equal to 0.05) increased DBC metabolism relative to control microsomes whereas DBC metabolism was not increased with 3MC-induced mouse liver microsomes. These data indicate that different enzymatic pathways are involved in the metabolic activation of DBC in the Hsd:ICR(Br) mouse and Sprague-Dawley rat.

Factors Affecting Carcinogenic Potential of Mixtures

Historically, exposure to complex mixtures such as soot, coal tar, mineral oils, and cigarette smoke has been associated with increased cancer mortality. Benzo[a]pyrene (BaP) has been used to predict the carcinogenic potency of mixtures. Two complete carcinogenicity C3H/HEJ mouse skin bioassays were undertaken to determine the effect of low doses of BaP on the carcinogenic potential of mixtures. A toluene solution containing 0.1% each of five noncarcinogenic polycyclic aromatic hydrocarbons (PAHs), anthracene, chrysene, pyrene, fluoroanthene, and phenanthrene, produced tumors in 23% of the mice with a latent period of 73 weeks. With the addition of a 0.001% BaP to the above solution, 47% of the mice produced tumors with a latent period of 66 weeks. In the second study, coal tar in toluene, which was determined to contain 0.0006% BaP, produced tumors in 51% of mice with a latent period of 73 weeks. In both studies the BaP solutions by themselves did not produce tumors. In a third study, the 9-, 2-, and 3-methylbenz[a]-anthracene compounds were noncarcinogenic using toluene as the solvent. With the substitution of n-dodecane for toluene all three compounds produced significant numbers of tumors. The results indicate that (1) low dose levels of BaP can have an impact on the carcinogenic potential of mixtures, (2) the presence or absence of BaP is not always sufficient to account for the observed potency and the synergistic effects of other substances which might be present, and (3) that certain noncarcinogenic methylbenz[a]anthracenes can have their carcinogenic potential altered by a change in the solvent used.

Influence of particle dose on the cytotoxicity of hamster and rat pulmonary alveolar macrophage in vitro

Silica and ferric oxide are common industrial exposures. Studies have indicated that all commonly occurring forms of crystalline silica can cause fibrotic lung disease. There is evidence to indicate that crystalline silica is carcinogenic in humans who have not developed silicosis, while amorphous silica is not carcinogenic in humans. An important biological response to particles deposited deep in the lung is their engulfment by pulmonary alveolar macrophages (AM). To assess the role of AM in silica-induced lung disease, particle size distribution and surface area of crystalline, gelled, precipitated, and fumed silica, ferric oxide, and aluminum oxide were characterized; the cytotoxicity of the particles to hamster and rat AM in vitro was measured at 0.0-0.5 mg/1 x 10(6) cells at 24 and 48 h using dye exclusion procedures. The count medium diameter for aluminum oxide, ferric oxide, and amorphous silica was equal to or less than 0.38 microns, while for crystalline silica the value was 0.83 microns. The surface areas for the amorphous silicas and the aluminum oxide ranged from 253 to 125 m2/g with gelled silica having the highest value; the values for crystalline silica and ferric oxide were 4.3 and 10.8 m2/g, respectively. Crystalline silica (1.6%) was detected in the fumed silica, while none was detected in precipitated or gelled silica. With gelled silica, based on the dose of the particle, the viability of the hamster AM decreased to 27% at 0.05 mg and to zero at 0.1 mg at 24 h. At doses of 0.05 and 0.1 mg of crystalline, precipitated, or fumed silica, the percent viability decreased significantly to 76-67% and 51-42%, respectively, and to zero at 0.5 mg. Macrophages viable at 24 h decreased further at 48 h compared with the control culture. The ferric oxide and the aluminum oxide showed minimal to no changes in viability. Similar results for the particles were obtained with rat AM. The results indicate that precipitated and fumed amorphous silica tested at equivalent doses are equally as toxic to AM lavaged from two species of rodents as crystalline silica; gelled silica is more toxic than crystalline. Ferric oxide and aluminum oxide are noncytotoxic in this system. The results of this study indicate that the dose as well as the surface area and surface characterization are important determinants in the cytotoxicity of hamster and rat AM to these particles.

Influence of the dose levels of cocarcinogen ferric oxide on the metabolism of benzo[a]pyrene by pulmonary alveolar macrophages in suspension culture.

The concurrent administration of a cocarcinogenic carrier particle such as ferric oxide (Fe2O3) and the polycyclic aromatic hydrocarbon lung carcinogen benzo[a]pyrene (BaP) results in a decreased latency and an increased incidence in the production of lung tumors in hamsters compared to the administration of BaP alone. The pulmonary alveolar macrophage (AM), the primary lung defense cell, has been shown to endocytize BaP, metabolize BaP to a more biologically active form, and then release the metabolites. Therefore, a study was undertaken to determine in a dose-response manner the effect of AM phagocytosis of a carrier particle (Fe2O3) on the metabolism of a carcinogen (BaP) and on the production of reactive oxygen. The AM were lavaged from hamsters and cultured in suspension (2.5 x 10(6) cells/vial) with BaP (62.5 nmol, 14C labeled) alone or adsorbed onto 0.5, 1.0, or 2.0 mg Fe2O3 in the presence of cytochrome c. Following separate ethyl acetate extractions of the AM and medium, the metabolites were isolated by high-performance liquid chromatography (HPLC) and quantified by liquid scintillation spectrometry. The production of superoxide anions was monitored by the reduction of cytochrome c. Concurrent exposure of AM to BaP-coated Fe2O3 resulted in a significant increase in the amount of BaP metabolites and superoxide anions produced with dose of Fe2O3. The following metabolites were identified in both the medium and the AM: 9,10-dihydrodiol, 7,8-dihydrodiol, 4,5-dihydrodiol, 9-hydroxy, 3-hydroxy, and 3,6-quinone. In general, the 7,8-dihydrodiol, which is considered to be the precursor of the ultimate carcinogenic metabolite of BaP, and superoxide anions, which have been shown to produce localized lipid peroxidation and edema in vivo, were significantly enhanced (p = .05, Duncans multiple comparison test) in AM exposed to all doses of Fe2O3 when compared to AM exposed to BaP alone. This Fe2O3 dose-related enhancement of superoxide anion production is indicative of increased endocytic capacity resulting in a greater amount of total metabolites being produced, in particular, the dihydrodiols of BaP, which are considered to be products of the active metabolic pathway of BaP.