Carcinogen metabolism in human airway cells

Afiatoxin B₁ (AFB₁) and the N-nitrosamines, N-nitrosodimethylamine (NDMA) and 4-(methyinitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are suspected lung carcinogens. These studies examine potential risks from pulmonary AFB₁ exposure as well as the modulation of CYP 2E1-mediated NNK and NDMA activation by nicotine, cotinine, and an aqueous cigarette tar extract (ACTE).; To study the ability of human lung cells to activate AFB₁, several human lung cell lines were used in culture: BEAS-2B, B-CMV1A2, B3A4, and normal human human bronchial epithelial (NHBE) cells.; NHBE expressed cytochromes P450 (CYPs) 1A2 and 3A4, and these isoforms were induced with 3-methylcholanthrene (3MC) exposure. This induction increased activation and DNA binding of AFB₁. Glutathione S-transferase (GST) activity was also induced by 3MC exposure, although no AFB₁-relevant activity was generated. These cells also lacked the AFB₁-detoxifying M1a-1a isoform.; BEAS-2B, B-CMV1A2, and B3A4 cells were used to study the roles of CYPs 1A2 and 3A4 in AFB₁ activation. BEAS-2B cells are an SV40 immortalized cell fine derived from NHBE cells. B-CMV1A2 and B3A4 cells are transfected with CYPs 1A2 and 3A4 and express these isoforms in high amounts. AFB1-DNA adducts and AFB₁ sensitivity were most significant in B-CMV1A2 cells. However, B3A4 cells suffered significant cytotoxicity at low AFB ₁ concentrations. Competitive quantitative RT-PCR showed that B-CMV1A2 cells expressed 6-fold more CYP 1A2 than the amount of CYP 3A4 expressed in B3A4 cells. When AFB₁-DNA adduct formation was adjusted for CYP content, 1A2 was the most efficient AFB₁ activator at low AFB ₁ concentrations (0–1.5 μM). GST activity was not significantly different among these cell lines.; The BEAS-2B cell types constitutively expressed p53. In the CYP-expressing BEAS-2B cells, p53 expression was inhibited by AFB₁ exposure and was associated with an increase in MDM2 expression. No p21WAF1 protein could be detected in AFB₁-exposed B-CMV1A2 or B3A4. Furthermore, no DNA ladder formation was detected.; To examine the modulation of N-nitrosamine activation by nicotine, cotinine, and ACTE, an in vitro assay was used to measure CYP 2E1 activity, and by adding nicotine, cotinine, and an ACTE to the reaction mixes, an inhibition of 2E1 activity was detected.