Inhibition of Benzo(a)pyrene-induced Lung Tumorigenesis in A/J Mice by Dietary N-Acetylcysteine Conjugates of Benzyl and Phenethyl Isothiocyanates during the Postinitiation Phase Is Associated with Activation of Mitogen-activated Protein Kinases and p53 Activity and Induction of Apoptosis

Recent studies in cell culture have shown that isothiocyanates (ITCs) induce apoptosis via activation of mitogen-activated protein (MAP) kinases and p53 pathways, suggesting a potential for ITCs or their conjugates to inhibit tumorigenesis during the postinitiation phase. To evaluate whether ITC compounds administered after carcinogen treatment inhibit lung tumorigenesis, we investigated in A/J mice the effects of the N -acetylcysteine ( N AC) conjugates of benzyl (BITC- N AC) and phenethyl ITC (PEITC- N AC) in the diet (15 μmol/g) administered after a single dose of 20 μmol benzo( a )pyrene [B( a )P]. The formation of lung adenomas was examined 140 days after B( a )P dosing. Both the BITC- N AC and PEITC- N AC-treated groups showed a significant reduction in lung tumor multiplicity from 6.1 ± 3.1 tumors/mouse in the B( a )P group fed the control diet to 3.7 ± 2.9 and 3.4 ± 2.7 tumors/mouse ( P = 0.018 and 0.006, respectively). To investigate the mechanisms of tumor inhibition, lung tissues were obtained at 21, 84, and 140 days at interim sacrifices during the bioassay. These tissues showed a significant increase in apoptosis as determined by in situ end-labeling for both ITC- N AC-treated groups. The MAP kinase pathway was activated in the ITC- N AC-treated groups. The activation of c-Jun NH 2 -terminal kinase was higher in the BITC- N AC and PEITC- N AC groups when compared with B( a )P-treated control. The phosphorylation of p38 and extracellular signal-regulated kinases (ErKs) 1 and 2 was also induced by these treatments. To determine the downstream target of MAP kinases, activator protein-1 (AP-1) and nuclear factor-κB activities were evaluated by gel shift assay. The AP-1 binding activity was remarkably increased in lung tissue from both the BITC- N AC and PEITC- N AC groups. No change in nuclear factor-κB binding activity was found, however. Phosphorylation of p53 was also higher than the constitutive levels in both ITC- N AC-treated groups, but no induction of p53 expression was detected. This study demonstrates the chemopreventive efficacy of the N AC conjugates of PEITC and BITC administered in the diet after a single dose of B( a )P for lung tumorigenesis and provides the first in vivo evidence that activation of MAP kinases, AP-1 transcription factors, p53 phosphorylation, and the induction of apoptosis may be involved in the chemopreventive activity of these compounds.