Effects of benzo[a]pyrene as an environmental pollutant and two natural antioxidants on biomarkers of reproductive dysfunction in male rats

Benzo[a]pyrene (B[a]P) is an environmental toxicant and endocrine disruptor. Therefore, the aim of the present study was to investigate the toxicity of B[a]P in testis of rats and also to study the role of silymarin and thymoquinone (TQ) as natural antioxidants in the alleviation of such toxicity. Data of the present study showed that levels of testosterone, estrogen and progesterone were significantly decreased after treatment of rats with B[a]P. In addition, B[a]P caused downregulation of the expressions of steroidogenic enzymes including CYP17A1 and CP19A1, and decreased the activity of 17-β hydroxysteroid dehydrogenase (17β-HSD). Moreover, B[a]P decreased the activities of antioxidant enzymes including catalase (CAT), glutathione peroxidase (GP_X) and superoxide dismutase (SOD), and significantly increased free radicals levels in testis of male rats. However, pretreatment of rats with silymarin prior to administration of B[a]P was found to restore the level of free radicals, antioxidant status, and activities of steroidogenic enzymes to their normal levels in testicular tissues. Moreover, histopathological finding showed that silymarin recovered the abnormalities occurred in tubules caused by B[a] P in testis of rats. On the other hand, TQ showed pro-oxidant effects and did not ameliorate the toxic effects of B[a] P on the testicular tissue since it decreased antioxidant enzymes activities and inhibited the protein expression of CYP11A1 and CYP21A2 compared to control rats. Moreover, TQ decreased the levels of testosterone, estrogen, and progesterone either in the presence or absence of B[a]P. It is concluded that B[a]P decreased testosterone levels, inhibited antioxidant enzymes activities, caused downregulation of CYP isozymes involved in steroidogenesis, and increased free radical levels in testis. Moreover, silymarin was more effective than TQ in restoring organism health and alleviating the deleterious effects caused by B[a]P in the testis of rats. Due to its negative impact, it is highly recommended to limit the use of TQ as a dietary supplement since millions of people in the Middle East are using it to improve their health.

     

Synthesis of zirconia-immobilized copper chelates for catalytic decomposition of hydrogen peroxide and the oxidation of polycyclic aromatic hydrocarbons

Chelating sorbents with diethylenetriaminepenta(methylene-phosphonic acid) (DTPMPA) and ethylenediaminetetraacetic acid ligands immobilized on zirconia matrix were prepared and subsequently saturated with Cu(II). All the Cu chelates catalyzed decomposition of H(2)O(2) yielding highly reactive hydroxyl radicals. All of them were also able to catalyze degradation of polycyclic aromatic hydrocarbons (anthracene, benzo[a]pyrene and benzo[b]fluoranthene). The most effective DTPMPA-based catalysts G-32 and G-35 (10 mg ml(-1) with 100 mmol H(2)O(2)) caused almost complete decomposition of 15 ppm anthracene and benzo[a]pyrene during a five day catalytic cycle at 30 degrees C. Anthracene-1,4-dione was the main product of anthracene oxidation by all catalysts. The catalysts were active in several cycles without regeneration.     

Rapid quantification of polycyclic aromatic hydrocarbons in hydroxypropyl-beta-cyclodextrin (HPCD) soil extracts by synchronous fluorescence spectroscopy (SFS)

Synchronous fluorescence spectroscopy (SFS) was directly applied to rapidly quantify selected polycyclic aromatic hydrocarbons (PAHs: benzo[a]pyrene and pyrene) in aqueous hydroxypropyl-beta-cyclodextrin (HPCD) soil extract solutions from a variety of aged contaminated soils containing four different PAHs. The method was optimized and validated. The results show that SFS can be used to analyse benzo[a]pyrene and pyrene in HPCD based soil extracts with high sensitivity and selectivity. The linear calibration ranges were 4.0x10(-6)-1.0x10(-3)mM for benzo[a]pyrene and 6.0x10(-6)-1.2x10(-3)mM for pyrene in 10mM HPCD aqueous solution alone. The detection limits according to the error propagation theory for benzo[a]pyrene and pyrene were 3.9x10(-6) and 5.4x10(-6)mM, respectively. A good agreement between SFS and HPLC was reached for both determinations of PAHs in HPCD alone and in soil HPCD extracts. Hence, SFS is a potential means to simplify the present non-exhaustive hydroxypropyl-beta-cyclodextrin (HPCD)-based extraction technique for the evaluation of PAH bioavailability in soil.     

Preliminary evidence of the role of hydrogen peroxide in the degradation of benzo[a]pyrene by a non-white rot fungus Fusarium solani

In order to study the enzymatic mechanisms involved in the successive steps of BaP degradation by a Deuteromycete fungus Fusarium solani, we developed an indirect approach by using inhibitors of enzymes. We used either specific inhibitors of peroxidases (i.e. salicylhydroxamic acid) and of cytochrome P-450 (i.e. piperonyl butoxyde) or inhibitors of both enzymes (i.e. potassium cyanide). Surprisingly, no expected decrease of BaP degradation was observed with most inhibitors tested. On the contrary, more BaP was degraded. Only butylated hydroxytoluene, which acts as a free radical scavenger, inhibited BaP degradation. The inhibition of these enzymes, which use H(2)O(2) as a cosubstrate, might have resulted in an increase of hydrogen peroxide availability in the fungal cultures. This enhancement could induce formation of reactive oxygen species (ROS) which might be the agents that initiate benzo[a]pyrene oxidation. This study proposed a hypothetic alternative metabolic pathway involved in PAH metabolism by Fusarium solani.     

Some Improvements in the Determination of Benzo [α] Pyrene in Air Samples

The earliest measurements of benz[α]pyrene (BαP) in air samples were almost exclusively based on chroma-tography on alumina columns followed by ultraviolet absorption, in spite of the fact that the original discovery and isolation of BαP in coal tar was based on its fluorescence properties. With the increasing use of fluorescence techniques, the vastly increased sensitivity over ultraviolet measurements has become generally apparent. In air sample extracts containing the four polycyclics, benzo[a]pyrene (BαP) benzo[k]-fluoranthene (BkF), benzo[g,h,i]perylene, (BghiP), and BαP, ultraviolet measurements for BαP are particularly, inaccurate. Fluorescence measurements of BαP are much more accurate, but some improvement in accuracy seems desirable. The present work deals with improvement of the fluorescence measurement of BαP. It now is possible to measure BαP in the presence of BkF with greatly increased accuracy. Interferences resulting from the presence of benzo[k]fluoranthene are now much lower.     

Removal of PAHs from water using an immature coal (leonardite)

It has been studied an immature coal (leonardite) as an adsorbent for removing PAHs [fluorene, pyrene, benzo(k)fluoranthene, benzo(a)pyrene and benzo(g,h,i)perylene] from water. To determine the efficiency of leonardite as an adsorbent of PAHs, factors such as pH, contact time and equilibrium sorption were evaluated in a series of batch experiments. There were no significant differences in the removal percentages for the various pH values studied, except for fluorene. The adsorption of fluorene was higher at lower pH values. The equilibrium time was reached at 24h. At this time, more than 82% of the pyrene, benzo(k)fluoranthene, benzo(a)pyrene and benzo(g,h,i)perylene had been removed. During the first 2h, the adsorption rate increased rapidly. After that time, however, there was a minor decrease. Equilibrium data were fitted to Freundlich models to determine the water-leonardite partitioning coefficient. Physical adsorption caused by the aromatic nature of the compounds was the main mechanism that governed the removal process. The polarity of the humic substances in leonardite may also have influenced the adsorption capacity.     

Benzo[a]pyrene degradation by Sphingomonas yanoikuyae JAR02

Batch experiments were conducted to characterize the degradation of benzo[a]pyrene, a representative high molecular weight (HMW) polycyclic aromatic hydrocarbon (PAH), by Sphingomonas yanoikuyae JAR02. Concentrations up to the solubility limit (1.2 microg l(-1)) of benzo[a]pyrene were completely removed from solution within 20 h when the bacterium was grown on salicylate. Additional experiments with [(14)C]7-benzo[a]pyrene demonstrated 3.8% mineralization over 7 days when salicylate was present is solution, and one major radio-labeled metabolite was observed that accounted for approximately 10% of the initial radio-label. Further characterization of the radio-labeled metabolite using HPLC/MS and HPLC/MS/MS identified radio-labeled pyrene-8-hydroxy-7-carboxylic acid and unlabeled pyrene-7-hydroxy-8-carboxylic acid as novel ring-cleavage metabolites, and a benzo[a]pyrene degradation pathway was proposed. Results indicate that biostimulation of HMW PAH degradation by salicylate, a water-soluble, non-toxic substrate, has significant potential for in situ bioremediation.     

Biomarker responses at different levels of biological organisation in crabs (Carcinus aestuarii) experimentally exposed to benzo(alpha)pyrene

The aim of this study was to validate a multi-trial biomarker approach for the evaluation of toxicological risk due to benzo(alpha)pyrene. Carcinus aestuarii, exposed to increasing concentrations of B(alpha)P in the water, was used as the bioindicator organism. A set of biomarkers were tested in order to: identify biological materials for biomarker and residue analysis; determine a group of sensitive techniques for the assessment of PAH contamination; investigate correlation between responses at different levels of biological organisation. The results underlined that BPMO activities in hepatopancreas and gills were a good biomarker of exposure to PAH-type compounds. B esterases activities in hemolymph and porphyrin patterns in excreta could be proposed as a non-destructive approach for evaluating chemical exposure in this species.     

Role of salicylic acid in alleviating oxidative damage in rice roots (Oryza sativa) subjected to cadmium stress

Time-dependent changes in enzymatic and non-enzymatic antioxidants, and lipid peroxidation were investigated in roots of rice (Oryza sativa) grown hydroponically with Cd, with or without pretreatment of salicylic acid (SA). Exposure to 50 microM Cd significantly decreased root growth, and activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), but increased the concentrations of H(2)O(2), malondialdehyde (MDA), ascorbic acid (AsA), glutathione (GSH) and non-protein thiols (NPT). However, pretreatment with 10 microM SA enhanced the activities of antioxidant enzymes and the concentrations of non-enzymatic antioxidants, but lowered the concentrations of H(2)O(2) and MDA in the Cd-stressed rice compared with the Cd treatment alone. Pretreatment with SA alleviated the Cd-induced inhibition of root growth. The results showed that pretreatment with SA enhanced the antioxidant defense activities in Cd-stressed rice, thus alleviating Cd-induced oxidative damage and enhancing Cd tolerance. The possible mechanism of SA-induced H(2)O(2) signaling in mediating Cd tolerance was discussed.     

Polycyclic aromatic hydrocarbons air levels in Florence,Italy, and their correlation with other air pollutants

Benzo(a)pyrene [B(a)P] air levels were measured in Florence (Italy) in the period 1992-2001. For the period 1999-2000 seven polycyclic aromatic hydrocarbons (PAH) (benzo(a)anthracene, crysene, benzo(a)pyrene (B(a)P), benzo(b)fluoranthene (B(b)F), benzo(k)fluoranthene, dibenzo(a,h)anthracene (DBA) and benzo(g,h,i)perylene (BGP)), were measured in the air in four different sites (one with heavy traffic (A), one in a park (B), one in a residential area (C) and one in a hill area (D)). B(a)P levels were elevated in 1992-1998 (maximum average value of winter months: 5.8 ng/ m3) but a decreasing trend was observed in the following years, probably due to improvement in vehicle emissions. The sum of PAH in the air in the period 1999-2000 was about one order of magnitude lower in the hill site (D) relative to the urban sites, and residential areas (B and C) had values 2.5-3 times lower compared to site A with a heavy traffic. PAH concentrations decreased in the warmer seasons of 2000 in all sites. A negative correlation was found between PAH levels and ozone. A positive correlation with carbon monoxide (CO) (r = 0.862, P < 0.001) and low B(a)P/BGP ratios, ranging from 0.44 to 0.51, indicated that vehicular traffic was the major PAH source in all monitored sites. Using B(a)P(TEF) values (toxic equivalency factors) for evaluating the biological activity of PAH, we found that the highest PAH contributors in terms of potential air carcinogenic activity were B(a)P and DBA. Therefore, in addition to B(a)P, DBA concentration should be considered in the evaluation of air quality in terms of PAH contamination.     

Mutagenic nitrated benzo[a]pyrene derivatives in the reaction product of benzo[a]pyrene in NO2-air in the presence of O3 or under photoirradiation

In order to clarify the contribution of nitrated products to the direct-mutagenic activity of products of the reactions of benzo[a]pyrene in NO2-air under various conditions, heterogeneous reactions of BaP deposited on filter in the air containing 10 ppm of NO2 have been conducted in dark or under photoirradiation. The reaction products have been analyzed by gas chromatography and mutagenicity of the products fractionated by preparative HPLC was assayed for Salmonella typhimurium strains TA98 and YG1024 in the absence of S9 mix. 3,6-dinitrobenzo[a]pyrene and 1,3-dinitrobenzo[a]pyrene, which are strong direct-acting mutagens, largely contributed to the total direct-acting mutagenicity of the dark reaction products in NO2-air. On the other hand, both the dark reaction in the presence of O3 and the photoreaction in NO2-air resulted in the formation of much smaller amounts of nitrobenzo[a]pyrenes than that observed in the dark reaction in the absence of O3. These results show that the contribution of other direct-acting mutagens to the total direct-acting mutagenicity of the products in these reactions should be considered. Benzo[a]pyrene lactones were identified in a highly mutagenic fraction of the products of the dark reaction in the presence of O3 and photoreaction and a nitrobenzo[a]pyrene lactone was also identified in a highly mutagenic fraction of the dark reaction products in the presence of O3. Nitrated oxygenated benzo[a]pyrene derivatives such as nitrobenzo[a]pyrene lactone were considered to largely contribute to direct-acting mutagenicity of the products of the dark reaction in the presence of O3 and photoreaction.     

Degradation mechanisms of benzo[a]pyrene and its accumulated metabolites by biodegradation combined with chemical oxidation

A high degradation extent of benzo[a]pyrene (BaP) should not be considered as the sole desirable criterion for the bioremediation of BaP-contaminated soils because some of its accumulated metabolites still have severe health risks to human. Two main metabolites of BaP, benzo[a]pyrene-1,6-quinone (BP1,6-quinone) and 3-hydroxybenzo[a]pyrene (3-OHBP) were identified by high performance liquid chromatography (HPLC) with standards. This study was the first time that degradation of both BaP and the two metabolites was carried out by chemical oxidation and biodegradation. Three main phases during the whole degradation process were proposed. Hydrogen peroxide-zinc (H(2)O(2)-Zn), the fungus - Aspergillus niger and the bacteria - Zoogloea sp. played an important role in the different phases. The degradation parameters of the system were also optimized, and the results showed that the effect of degradation was the best when fungus-bacteria combined with H(2)O(2)-Zn, the concentration range of BaP in the cultures was 30-120mg/l, the initial pH of the cultures was 6.0. However, as co-metabolites, phenanthrene significant inhibited the degradation of BaP. This combined degradation system compared with the conventional method of degradation by domestic fungus only, enhanced the degradation extent of BaP by more than 20% on the 12d. The highest accumulation of BP1,6-quinone and 3-OHBP were reduced by nearly 10% in the degradation experiments, which further proved that the combined degradation system was more effective as far as joint toxicity of BaP and its metabolites are concerned.     

Responses of antioxidant systems and LPO level to benzo(a)pyrene and benzo(k)fluoranthene in the haemolymph of the scallop Chlamys ferrari

The effects of benzo(a)pyrene (BaP), benzo(k)fluoranthene (BkF) and their mixture on antioxidant enzyme activities and lipid peroxidation (LPO) levels of haemolymph of scallop (Chlamys ferrari) were studied. The superoxide dismutase (SOD) activities of 0.5 microg/L and 1.0 microg/L were significantly higher than controls (P<0.05), while it increased at beginning and then dropped (lower than controls) in the end at 10.0 microg/L and 50.0 microg/L PAHs groups. The catalase (CAT) activities were very little during the whole experimental time. The glutathione peroxidase (GPx) activities in each PAHs group all increased significantly (P<0.05). LPO levels all increased significantly (P<0.05) with time at each PAHs group except for the 0.5 microg/L group of less than hour 12. The toxicity of PAHs in a descending order was BaP>BkF>mixture of BaP and BkF. The changes in antioxidant enzyme activities and LPO level in haemolymph could reflect the detoxification functions and damage levels of whole organism.     

Benzo[a]pyrene exposure influences the cardiac development and the expression of cardiovascular relative genes in zebrafish (Danio rerio) embryos

It is reported that the most abundant polycyclic aromatic hydrocarbons (PAHs) in weathered crude oils are cardiotoxic. However, the action mechanism of PAHs on vertebrate cardiovascular development and disease is unclear. In the present study, the cardiac morphology and functioning of zebrafish embryos exposed to benzo[a]pyrene [B(a)P], as a high-ring PAHs, for 72 h were observed and determined. The results showed that B(a)P exposure resulted in cardiac developmental defects in zebrafish embryos. Significant changes in expression level of multiple genes potentially critical for regulating the B(a)P-induced cardiovascular developmental defects were also found. A gene network regulating cardiac development perturbed by B(a)P exposure was identified and established by computational analysis and employment of some databases. The information from the network could provide a clue for further mechanistic studies explaining molecular events regulating B(a)P-mediated cardiovascular defects and consequences.     

Removal of pyrene and benzo(a)pyrene from contaminated water by sequential and simultaneous ozonation and biotreatment.

The removal of pyrene and benzo(a)pyrene from contaminated water by sequential and simultaneous ozonation-bioremediation techniques was investigated. During the sequential treatment, ozonation using 0.5 or 2.5 mg/L ozone was used as a pretreatment process, whereas, during the simultaneous treatment process, ozonation of hydrocarbon-contaminated water at a predetermined duration using 0.5 mg/L ozone was made in the presence of microbial biomass. Ozonation was not beneficial for the removal of pyrene. However, despite a decreased specific biodegradation rate, ozonation improved the overall elimination of benzo(a)pyrene during both treatment processes. The overall removal of benzo(a)pyrene increased from 23 to 91% after exposure of the water to 0.5 mg/L ozone for 30 minutes during the simultaneous treatment process and further to 100% following exposure to 2.5 mg/L ozone for 60 minutes during the sequential treatment mode, demonstrating the benefits of combined ozonation-biological treatment for the removal of polycyclic aromatic hydrocarbons.     

CMAQ simulations of the benzo(a)pyrene distribution over Europe for 2000 and 2001

In order to investigate benzo(a)pyrene (B(a)P) concentrations in ambient air in Europe and the respective deposition fields an expanded version of the Community Multiscale Air Quality (CMAQ) Modelling system has been used to simulate these fields for the years 2000 and 2001. Significant differences exist between different regions in Europe and between winter and summer concentrations. Modelled B(a)P concentrations are highest in Central Europe, South Ukraine and around Moscow. Wet deposition shows mainly the same regional distribution as the concentrations. Simulated concentrations and depositions are compared to measurements at selected sites of the EMEP network and of the German Federal Environmental Agencies. The measurements are on average underestimated by 50%. The temporal evolution and the regional distribution of B(a)P are captured by the model. In the approach presented here, particle bound B(a)P undergoes degradation by reactions with ozone. In order to assess the importance of the degradation process the results are compared to a model run where no degradation of particle bound B(a)P is considered. It was found that the inclusion of heterogeneous reactions of B(a)P with ozone results in a reduction of the modelled air concentration by approx. a factor of 5.     

Bioaccumulation of cyclopenta[cd]pyrene and benzo[ghi]fluoranthene by mussels transplanted in a coastal lagoon

The bioaccumulation of two isomeric non-alternant non-priority polycyclic aromatic hydrocarbons (PAHs), namely cyclopenta[cd]pyrene and benzo[ghi]fluoranthene, was investigated in caged mussels (Mytilus galloprovincialis) exposed for 30 days in three sites of a coastal lagoon (Pialassa Baiona, Ravenna, Italy) contaminated by pyrogenic PAHs. The concentration of cyclopenta[cd]pyrene and benzo[ghi]fluoranthene increased from undetectable levels in reference mussels withdrawn from the Adriatic sea to 10-30 ng g(-1) dry weight in transplanted mussels. Other contaminants bioaccumulated by caged mussels included pyrene, fluoranthene and mercury. Whilst the isomer concentration ratio pyrene/fluoranthene in biota was comparable to that observed in sediments, the cyclopenta[cd]pyrene/benzo[ghi]fluoranthene ratio was much lower in mussels than in sediments. The lower sediment biota accumulation factor of cyclopenta[cd]pyrene with respect to that of benzo[ghi]fluoranthene was tentatively attributed to the greater biological activity of the former compound, which contains a reactive olefinic bond in the cyclopenta fused ring moiety. Given the higher mutagenic activity of cyclopenta[cd]pyrene with respect to other priority PAHs, its bioaccumulation from contaminated sediments may rise considerably the overall toxicity of PAH residues in exposed biota.     

Alterations to proteome and tissue recovery responses in fish liver caused by a short-term combination treatment with cadmium and benzo[a]pyrene

The livers of soles (Solea senegalensis) injected with subacute doses of cadmium (Cd), benzo[a]pyrene (B[a]P), or their combination, were screened for alterations to cytosolic protein expression patterns, complemented by cytological and histological analyses. Cadmium and B[a]P, but not combined, induced hepatocyte apoptosis and Kupfer cell hyperplasia. Proteomics, however, suggested that apoptosis was triggered through distinct pathways. Cadmium and B[a]P caused upregulation of different anti-oxidative enzymes (peroxiredoxin and glutathione peroxidase, respectively) although co-exposure impaired induction. Similarly, apoptosis was inhibited by co-exposure, to which may have contributed a synergistic upregulation of tissue metalloproteinase inhibitor, β-actin and a lipid transport protein. The regulation factors of nine out of eleven identified proteins of different types revealed antagonistic or synergistic effects between Cd and B[a]P at the prospected doses after 24 h of exposure. The results indicate that co-exposure to Cd and B[a]P may enhance toxicity by impairing specific responses and not through cumulative damage.     

Effects of metabolites of benzo(a)pyrene on unschedule DNA synthesis in BALB/3T3 cell line

In order to explore the damage from metabolites of benzo(a)pyrene on DNA of mammalian cells, the effects of four metabolites of benzo(a)pyrene (anti-BPDE, syn-BPDE, 3-OH-BP and 9-OH-BP) on synthesis of DNA and unschedule DNA synthesis (UDS) in BALB/3T3 cells were assayed, by methods of single-labeling and double-labeling. The results showed that all of the four agents were able to increase the synthesis of DNA, but only three of them (apart from syn-BPDE) induced UDS in BALB/3T3 cells. The above indicates that the metabolites of benzo(a)pyrene are able to damage DNA in BALB/3T3 cells, and that this effect may be relative to the sterical structure of metabolites of benzo(a)pyrene.     

Metabolism of aromatic hydrocarbons by the filamentous fungus Cyclothyrium sp

The metabolism of biphenyl, naphthalene, anthracene, phenanthrene, pyrene and benzo[a]pyrene by Cyclothyrium sp. CBS 109850, a coelomycete isolated for the first time in Brazil from industrially polluted estuarine sediment, was studied. The metabolites were extracted and separated by high performance liquid chromatography (HPLC) and characterized by UV spectral analyses and mass, and proton nuclear magnetic resonance ((1)H NMR) spectrometry. Cyclothyrium sp. transformed biphenyl to 4-hydroxybiphenyl and anthracene to anthracene trans-1,2-dihydrodiol. This isolate metabolized 90% of [9-(14)C]phenanthrene, producing phenanthrene trans-9,10-dihydrodiol as a major metabolite, phenanthrene trans-3,4-dihydrodiol, 1-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, and a novel metabolite, 2-hydroxy-7-methoxyphenanthrene. Circular dichroism spectra analyses indicated that the major enantiomers of phenanthrene trans-9, 10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol and pyrene trans-4,5-dihydrodiol, a pyrene metabolite produced previously by Cyclothyrium sp. CBS 109850, were predominantly in the (R,R) configuration, revealing a high stereoselectivity for initial monooxygenation and enzymatic hydration of phenanthrene and pyrene by Cyclothyrium sp. CBS109850. The results also show a high regioselectivity since the K-regions of phenanthrene and pyrene were the major sites of metabolism.