With prevention in mind, researchers investigate how benzopyrene can cause cancer

Potent carcinogen and mutagen is present in cigarette smoke, automotive exhaust, burnt wood fumes and barbecued meat.

By Karina Toledo  |  Agência FAPESP – 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 FAPESP and coordinated by Ana Paula de Melo Loureiro, a professor in the University of São Paulo’s School of Pharmaceutical Sciences (FCF-USP) in Brazil.

Preliminary findings were disclosed in August 2017 during the Fifth Symposium on Epigenetics & Medical Epigenomics, held in São Paulo.

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.”

Part of the project was conducted by Tiago Franco de Oliveira, with support from FAPESP in the shape of a postdoctoral scholarship. The current stage, which involves supplementation with nicotinamide riboside, is the PhD project of Everson Willian Fialho Cordeiro, also with a scholarship from FAPESP.

Methodology

The experiments are being performed with normal human lung cells, more precisely with bronchial epithelial cells, which are incubated with benzo[a]pyrene for a week. Because the substance is absorbed rapidly and biotransformed, it has to be replenished in the cell cultures every day, Loureiro explained.

At the end of the incubation period, the cells are transferred to a semi-solid medium containing agarose, a polysaccharide obtained from algae, in order to prevent adhesion to the culture dish.

“We know a normal epithelial cell is unable to grow in this semi-solid medium without anchorage,” Loureiro said. “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].”

The transformation was observed in cultures exposed to 0.5 and 1 micromolar (μM) benzo[a]pyrene and grown for seven days. Cells exposed to 0.1 μM benzo[a]pyrene did not grow in agarose. According to Loureiro, lower concentrations of the carcinogen may induce cellular transformation after longer periods of exposure.

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.

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. The other procedures were similar to those of the previous test.

At the end, the cells exposed to the carcinogen and supplemented with nicotinamide riboside proved unable to grow in the agar medium, displaying similar behavior to the control cells, which were not exposed to benzo[a]pyrene.

“We know tumor cells have an altered metabolism designed for growth,” Loureiro said. “We now plan to investigate how supplementation with nicotinamide riboside protects against transformation of cells that are in contact with a substance known to be carcinogenic.”