Biological activity and environmental removal of Benzo(a)pyrene, the most carcinogenic polycyclic aromatic hydrocarbon


Danielle Fagnani
Drexel University
Chem 367
December 4, 2010

Abstract

Benzo(a)pyrene is an polycyclic aromatic hydrocarbon (PAH) that is one of the most well-known and potent carcinogens of its kind. Benzo(a)pyrene was discovered in the early 1900’s and more in-depth research of its biological activity continued for the rest of the century. BaP is a by-product of daily industrial and personal activities. This is a problem due to it’s ability to bind to DNA and cause genetic mutations that can cause cancer. The details of this interaction are included in this review. The activity of B(a)P in the human body cannot be reversed. Removing the pollutant from the environment can be done by various biodegradable and chemical methods discussed in this paper.

Background of Benzo(a)pyrene


What is Benzo(a)Pyrene

Benzo(a)pyrene (BaP) is a five-membered hydrocarbon that has been widely studied for it carcinogenic effects. The compound is part of a class of compounds known as Polycyclic Aromatic Hydrocarbons (PAHs), which consist of at least three fused benzene rings (10). BaP is considered a high molecular weight BaP and is not water soluble. PAHs usually occur as complex mixtures containing different configurations of fused rings that lead to different properties (7,10,12). PAHs are formed by the incomplete combustion of organic material and can be found in many various places. Benzo(a)pyrene and PAHs in general are known for their carcinogenic and mutagenic properties.


B(a)P
Fig.1 Benzo(a)pyrene shown with numbered carbons. Bay region and K region are two sections of the compound where diol epoxides can form.



Discovery
Interest in PAHs was sparked in the nineteenth century when the high occurance of skin cancer was found in paraffin refining, shale oil, and coal tar industry employees. In 1922 Ernest L. Kennaway, a British scientist, began experimentation on coal tar that lead to the discovery and characterization of Benzo(a)pyrene. Other scientists joined his efforts over the next decades to purify and identify the compound. E.L. Kennaway and I. Hieger published a paper in 1930 in The British Medical Journal titled Carcinogenic substances and their fluorescence spectra that detailed their experimentation and progress in identifying the carcinogen in coal tar (2). In the next years, other scientists joined his efforts and followed his procedures.The team of scientists compared the fluorescence of PAHs purified from coal with synthesized standards. Those that matched up were able to produce tumors on mice. C.L. Hewett and J.W. Cook were able to synthesize BaP and properly identify Benzo(a)Pyrene as the major component of carcingenic PAHs in coal tar. In 1939 all of the scientists involved (Kennaway, Mayneord, Hewett, Hieger, and cook) won the first Anna Fuller Memorial prize for their accomplishments in cancer research. Experimentation then moved into finding the detailed pathway that benzo(a)pyrene followed to cause cancer (1).

Sources of Benzo(a)Pyrene


Formation from combustion
BaP as well as other PAHs are produced by the incomplete combustion or high-temperature processes of organic material. PAHs can also be the the resulting product of the pyrolysis,the thermochemical decomposition of organic material at elevated temperatures in the absence of oxygen, of organic matter . BaP forms between the 300 and 600 degrees celcius.There are multiple theories that describe the mechanism of PAH formation. Pathways that utilize free radicals has been researched and deemed to be the most important (13). A general reaction scheme for the formation of PAHs was developed by Bockhorne et al in 1983 (22).



formation_of_PAHs.JPG
Fig. 2 Reaction scheme for the formation of aromatic hydrocarbons. (image from (16), originally from (25).




Where to find BaP
BaP is exposed to human everyday and can be found in the atmosphere, soil, waterways, oceans, and the food chain (14). Natural combustion processes that lead to the formation BaP and other PAHs include volcanic eruptions and forrest fires. However, most BaP is produced by anthropogenic process that vary from industrial, chemical, transportation, food preparation and waste removal combustive processes (12). Industrial processes that involve the use of coal tar, asphalt, coal, petroleum, aluminum and soot production. The burning of any sort of organic material is likely to release a great mixture of PAHs including BaP. Wood stoves, fire places, and furnaces in homes and buildings is a producing source. The organic chemistry industry of the early 20th century began using and releasing PAHs in a wide variety of applications. Some PAHs were used as a building block in the synthesis of dyes. Motorized vehicle exhaust and the burning of fuels are a contributing source of PAHs in the environment. Common food preparation processes produce BaP, this includes char-boiling, grilling, roasting, frying, or baking. The main source is from smoked and grilled meat, poultry and fish. Levels as high as 200 μg/kg food have been found for individual PAH in smoked fish and meat (9). BaP partitions mainly into soil (82%) and sediment (17%) (9). Soil contamination coupled with air contamination lead to deposits can be found on fresh vegatables, fruit, wheat and other crops, however, this is more common near industrial sources. Incineration is a thermal treatment of waste removal that combusts waste that is composed of organic material. This process often produces BaP and hazardous products that are found in the resulting ash or released into the atmosphere (14). Another source of BaP is from the smoke released from tobacco and marijuana (3).

BaP sources in air can deposit onto soil and water. Bioaccumalation is the accumlation of a substance in various tissues of living organism. BaP tends to accumulate in the lipid tissue of plants and animals. BaP in water supplies will accumluate in marine organism due to the lipophilic nature of BaP. A study done by a research group in Nigeria used a GC/MS method to analyze BaP concentration is fish samples from the Niger Delta (12). This teams research show a range of 1.5 μg/kg - 10.2 μg/kg concentration of BaP in fish, with levels varying as a function of water contamination.The bioaccumulation of BaP in marine organisms can disperse throughout the foodchain, which is responsible for approximately 97% of human exposure to BaP (15).

BaP has can be found as a contaminent in many aqueous matrices including surface waters, seawaters, groundwater, drinking water, as well as in sediments (8). Drinking water can become contaminated by leaching from linings of water storage tanks and distribution lines. The Unites States Environmental Protection Agency set the maximum contaminant level goal (MCLG) and the minimal contaminant level (MCL) of BaP in drinking water at zero and 0.0002 mg/L respectively (16).

Human Health Effects of BaP: Carcinogenesis



Mechanism of cancer-causing DNA mutation
BaP is one of the most carcinogenic PAHs. BaP can get into the human body through inhalation, skin absorption, and ingestion through diet. The carcinogenic effects of BaP have been tested widely in animals (1). BaP will cause skin irritation and the development of tumors when applied to the skin of a mouse. These test results coupled with statistics of cancer-occurance in humans exposed to BaP prove the high carcinogenic activity of BaP (18). Benzo(a)pyrene causes cancer by binding with DNA which induces cancer-causing gene mutations. BaP must first be converted into the metabolite, an intermediate or product of metabolism, that intercalates between two starnds of DNA. Intercalation is the reversible inclusion of a molecule between two other molecules. BaP is metabolized into it's epoxide formation by enzymes of the cytochrome P450(CYP) super family (5). Cytochrome P450 (CYP) is a mixture of enzymes present in human and animal tissues that function to catalyze the oxidation of organic substances. These enzymes metabolically activate BaP into a reactive metabolite, a bay region diol epoxide (BaPDE). The bay region is the angle formed between benzo rings formed in a nonlinear arrangement, in the case of BaP the bay region is borded by the region between carbon 10 and 11 (fig. 1). BaP is oxidized by monooxygenases (MO) to BaP-7,8-oxides, followed by epoxide hydrolases (EH) to BaP-7,8-dihydrodiol, and then finally oxidized by MO into BaP-7,8-diol-9,10-epoxide. The oxidation of BaP leads to four isomers, the most active one in producing tumors is (+)-anti-BaP-7,8-diol-9,10-epoxide ((+)-anti-BaPDE), (shown in fig.3) This form is the most tumorigenic and intercalates with DNA (6).


BaPDE_pathway_(nature).JPG
Fig. 3 "Major pathway of metabolic activation of BaP. The biotransformations are catalyzed by MO (monooxygenase); EH (epoxide hydrolase). the structure of the major BaPDE-guanine adduct in DNA is also shown. (1)




Once in BaP is converted to BaPDE and intercalated with DNA it can form a DNA adduct, which is a piece of DNA covalently bonded to a cancer-causing molecule, leading start of a cancerous cell (carcinogenesis). The adduct can cause miscoding that can induce harmful gene mutations. These mutationt can cause cancer if they occur is oncogenes, tumor supressor genes, and other genes involved in cell-cycle regulation. BaP convalently bonds with DNA after intercalation by the nucleophilic attack on the (+)-anti-BaPDE epoxide by the exocyclic NH2 group of either adenine or guanine (6). BaP most commonly reacts with the N2 position of guanine, but also reacts in the N6 position of adenine which interrupts the guanine-cytosine (GC) and adenine-thymine (AT) base pairs respectively. The main adduct ( BaPDE-N2-dG, fig 3) is formed between the 10-carbon on BaPDE and the 2-amino group of guanine. The reasoning behind this interaction may be the more stable thermodynamics of BaPDE-N2-dG formation (6). This DNA adduct can be hydrolized to BaP-tetraol, which is the substance researchers can use when measuring BaP-DNA adduct levels, however there a number of different quantitative methods to determine this (17).

Recovery and Removal



Benzo(a)pyrene is a carcinogenic pollutant in the environment. There has been a substantial amount of research conducted on removal of BaP. Many natural methods have been discovered as well as the development of chemical and physical methods. Bioremidation of contaminated areas can be done by natural or chemical methods. Natural methods are part the biodegredation field and is becoming a more popular method of chemical contamination removal. This method utilizes a biological means of contaminent destruction (4). This is generally a greener alternative to physical and chemical means.

Biodegradation of PAHs can occur under aerobic & sometimes anarobic conditions. Anaerobic conditions are generally much slower and unfeasible for BaP since it does not occur on PAHs that contain more than three aromatic rings. Under aerobic conditions, BaP and other PAHs are degraded in three steps. First is the transformation into a central intermediate, followed by activation and cleavage of the aromatic ring, and then conversion of the hydrocarbon chain into central metabolites (19).

A variety of species in aerobic conditions are able to oxidize BaP. Many bacteria species have shown the ability to degrade BaP. When exposed to Sphingomonus paucimobilis, Bap concentration decresed 5%. This occured by hyroxylation followed by the ring cleavage of the 7,8,9,10-benzo-ring, which evolves carbon dioxide gas. BaP can be degraded by many other strain of bacteria. Many other bacterial strains which have shown must faster rates of degrading BaP that is mixed into a complex of PAHs. Some bacteria overgrow in highly contaminated areas from excessive degrading and feeding off of PAHs (19). Algea is another biological tool for biodegradation and behaves co-metabolically with bacteria. Algae can convert BaP to diols and quinones if 5-6 days. Many fungi species can degrade PAHs. White-rot fungi (WRF) is a widely study fungi in the degredation of Bap. WRF is a family of fungi that have many environemntal advantages when used in bioremediation. "The very non specific nature of the mechanisms used by these fungi allows them to degrade even complex mixtures of pollutants all the way to carbon dioxide" (20). The lignin-degrading system of WRF degrades a wide variety of pollutants. The lignolytic enzymes used for oxidation are lignin peroxidase, laccase, and manganese peroxidase. These enzymes, as well as similar enzymes in other species, degrade BaP by oxidation. BaP, like other PAHS can sometimes become adsorbed onto soil sediments, making it harder to be biodegraded. A augmented method of biodegration can be used to combat this. The addition of biosurfactant-producing bacteria and light oils can serve to increase the bioavailability of molecules and metabolic potential of the bacterial community.

The chemical methods used to degrade PAHs mostly utilize volatilization and oxidation. Fenton's method is an example of an Advanced Oxidation Process (AOP) that is used for the degradation of BaP in aquesous matrices (8). Fentons method consists of two steps: Fenton's oxidation, which is the formation of hydroxyl radicals, followed by Fenton's coagulation, which is ferris coagulation (21). Fenton's oxidation begins with an aqueous solution of hydrogen peroxide and iron to form the hydroxyl radical and iron(III) ion [fig. 4, line (1)]. These then coagulate which serves to oxidize BaP [fig.4, line (4)]. A schematic of hydroxyl radical oxidation is scene in fig. 4 (21). Fenton's treatment is proven to be an appropriate method to completely degrade BaP in an aqueous environment. Fenton's treatment has been shown to work synergystically with biodegredation methods (8).


Fenton's_Treatment.JPG
Fig. 4 General reaction scheme of the oxidation of organic compunds by Fenton's Treatment, (24)


Conclusion


B(a)P is a carcinogen and a pollutant. It is product of incomplete combustion of organic material and can be released from many sources, including industrial emissions, fuel exhaust, smoking, and food preparation. It deposits mostly in soil and sediment and due to its hydrophobic nature has the tendency to bioaccumulate in living organism and enter the food chain. It causes cancer in the human body by its specific conversion to a diol epoxide metabolite and formation a DNA adduct. There is still research efforts dedicated to finding detailed pathway BaPDE-DNA adducts use to cause very specific cancers. Bioremidation is the best to limit human exposure. BaP can be biodegraded by the aerobic oxidation from certain bacteria, alges, and fungi, specifically White-rot-fungi. The best chemical method to remove BaP is the Fenton’s method, an Advanced Oxidation Process.


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