Differential activity of NO synthase inhibitors as chemopreventive agents in a primary rat tracheal epithelial cell transformation system

Abstract

A model to study the effectiveness of potential chemopreventive agents that inhibit neoplastic process by different mechanisms has been used to test the efficacy of seven nitric oxide synthase (NOS) inhibitors. Five selective inducible NOS (iNOS) inhibitors: S-methyl isothiourea (S-MITU), S-2-aminoethyl isothiourea (S-2-AEITU), S-ethyl isothiourea (S-EITU), aminoguanidine (AG), 2-amino-4-methyl pyridine (2-AMP), and two non selective general NOS inhibitors: l-N(6)-(1-iminoethyl) lysine (IEL) and N(omega)-nitro-l-arginine (NNLA), were tested for efficacy against a carcinogen, benzo[a]pyrene (B[a]P)-induced primary rat tracheal epithelial (RTE) cell transformation assay. RTE cells were treated with B[a]P alone or with five nontoxic concentrations of an NOS inhibitor and the resulting foci at the end of 30 days were scored for inhibition of transformation. The results indicate that all three isothiourea compounds inhibited B[a]P-induced RTE foci in a dose-dependent manner. S-AEITU was the most effective inhibitor with an IC(50) (the molar concentration that inhibits transformation by 50%) of 9.1 microM and 100% inhibition at the highest dose tested (30 microM). However, both S-EITU and S-MITU showed a maximum percent inhibition of 81% and 100% at 1 mM with an IC(50) of 84 and 110 microM, respectively. 2-AMP did not show any dose-dependent response, but was highly effective (57% inhibition) at an intermediate dose of 30 microM and an IC(50) of 25 microM. Similar to thiourea compounds, AG exhibited good dose-dependent inhibition with a maximum inhibition of 86% at 1 mM. NNLA and IEL were negative in this assay. Based on the IC(50) values, NOS inhibitors were rated for efficacy from high to low as follows: S-2-AEITU<2-AMP<AG<S-MITU<S-EITU. The data from this study identify NOS inhibitors as a novel class of chemopreventive agents that can be developed for lung cancer prevention.

Figure 1

Efficacy of thiourea class of iNOS inhibitors in the RTE transformation assay. Primary RTE cells were treated with B[a]P alone or with five half-log concentrations of each thiourea compound. Transformed colonies (type II+III foci) were scored at the end of 30 days and the results are expressed as percent inhibition of transformation in compound-treated versus untreated B[a]P control.

Figure 2

Efficacy of other iNOS inhibitors in the RTE transformation assay. Primary RTE cells were exposed to B[a]P alone or with various concentrations of AG and 2-AMP and the number of transformed colonies (type II+III foci) after 30 days were scored. The results are expressed as percent inhibition of transformation in iNOS-treated versus B[a]P control.

Figure 3

Negative response of nonselective NOS inhibitors in the RTE transformation assay. An agent is considered negative if there is less than 20% inhibition of B[a]P-induced transformation.