Benzo[a]pyrene is a potent carcinogen and mutagen present in cigarette smoke, automotive exhaust, burnt wood fumes, barbecued and smoked meat etc.
Understanding the mechanisms whereby benzo[a]pyrene, which belongs to the class of polycyclic aromatic hydrocarbons (PAHs), may induce malignant transformation of human cells is the aim of a research project supported by the São Paulo Research Foundation (FAPESP) and coordinated by Ana Paula de Melo Loureiro, a professor at the University of São Paulo's School of Pharmaceutical Sciences (FCF-USP) in Brazil.
According to Loureiro, the idea is to identify the cellular pathways or sequences of biological reactions involved in the development of cancer and hence find possible targets for prevention or treatment of the disease.
"Tests have shown that supplementing cell cultures with nicotinamide riboside, one of the components of vitamin B3, protects cells and prevents malignant transformation," she said. "We now want to find out exactly how this happens and whether the compound in question can be used in chemoprevention."
A substance intensifies cell metabolism
In experiments performed with regular lung cells – more precisely bronchial epithelial cells – cultures were exposed to 0.5 and 1 micromolar (μM) benzo[a]pyrene for seven days.
Recent data in the scientific literature suggest that the appearance of tumors is closely associated with genetic alterations and also with epigenetic alterations, which may activate the expression of protumoral genes or silence protective genes, for example.
What most drew the scientists' attention, however, was a significant drop in the levels of metabolites involved in the production of energy for cells shortly after the first hour of exposure to benzo[a]pyrene. Throughout the exposure period, the cells readapted metabolically, and by the end of the period, the levels of metabolites had risen in exposed cells compared with the control group.
"It was for this reason that we had the idea of supplementing the cultures with nicotinamide riboside, a precursor to nicotinamide adenine dinucleotide [NAD+], which is essential to cellular metabolism and to the production of ATP [adenosine triphosphate, a molecule that stores energy for cell use]," Loureiro said.
Supplementation with nicotinamide riboside (1 μM) began 24 hours before exposure to benzo[a]pyrene and was renewed daily with the latter. Because benzo[a]pyrene is absorbed rapidly and biotransformed, it has to be replenished in the cell cultures every day, Loureiro explained.
Understanding the connection between benzo[a]pyrene and tumoral development
At the end of the seven-day period, the cells were transferred to a semi-solid medium containing agarose, a polysaccharide obtained from algae, in order to prevent adhesion to the culture dish. The researcher says that a normal epithelial cell is unable to grow in this semi-solid medium without Anchorage.
"To make this possible, the expression of a number of genes and proteins must be altered so as to favor tumor development, for example by silencing the expression of cadherins [calcium-dependent adhesion molecules that form junctions to bind nearby cells together]," said Loureiro.
Analysis performed during the incubation period showed the occurrence of DNA alterations – both genetic (lesions that caused mutations in the nucleotide sequence) and epigenetic (increased levels of 5-methylcytosine, which alters gene expression). Cells grown in the semi-solid medium displayed global hypomethylation (reduction in levels of 5-methylcytosine), a characteristic of tumor cells.
Supplementing cells exposed to the carcinogen nicotinamide riboside yielded positive results since they proved unable to grow in the agar medium, displaying similar behavior to the control cells, which were not exposed to benzo[a]pyrene.
About São Paulo Research Foundation (FAPESP)
The São Paulo Research Foundation (FAPESP) is a public institution with the mission of supporting scientific research in all fields of knowledge by awarding scholarships, fellowships and grants to investigators linked with higher education and research institutions in the State of São Paulo, Brazil. FAPESP is aware that the very best research can only be done by working with the best researchers internationally. Therefore, it has established partnerships with funding agencies, higher education, private companies, and research organizations in other countries known for the quality of their research and has been encouraging scientists funded by its grants to further develop their international collaboration. For more information: http://www.fapesp.br/en.