Chemoprotection by natural chlorophylls in vivo: inhibition of dibenzo[a,l]pyrene-DNA adducts in rainbow trout liver

Abstract

Naturally occurring chlorophylls (Chl) have shown anti-mutagenic activity but little is known about their chemoprotective properties in vivo. This study examined the effect of Chl on formation in vivo of DNA adducts by the potent environmental carcinogen dibenzo[a,l]pyrene (DBP), using rainbow trout as the animal model. Fingerling trout were fed diets containing 200 p.p.m. DBP alone or with one of the following preparations incorporated at 3000 p.p.m. total chlorins: purified pheophytin a (Phe a) (94%); semi-purified Chl a (77%, 23% Phe a), commercial Chl a (88%, 12% other Chl a-related compounds); crude spinach extract (53% Chl a, 19% Chl b, 14% Phe a, 9% carotenoids); commercial Cu-chlorophyllin (55% chlorins, 45% neutral salts), as a known inhibitory control. After 2 weeks dietary treatment, the animals were killed and organs were collected. Stable DBP-DNA adducts from liver were quantified after 33P-post-labeling and separation by reversed-phase HPLC. Total DBP-DNA adducts in the DBP-only group were 2.46 +/- 0.32 adducts/10(6) nucleotides. All chlorophyll treatment groups showed significantly lower adduct levels (P < 0.001, Tukey's HSD test), as follows: crude spinach extract, 0.64 +/- 0.14; semi-pure Chl a, 0.5 +/- 0.11; commercial Chl a, 1.26 +/- 0.17; Phe a, 0.95 +/- 0.01; chlorophyllin, 0.78 +/- 0.09. The various treatments suppressed DBP-DNA adducts essentially uniformly across the HPLC profile, which is consistent with complex formation and reduced carcinogen uptake as the predominant protective mechanism. The chlorophyll-mediated reduction in DBP-DNA adducts in vivo is the first demonstration of anti-genotoxic activity of these common dietary phytochemicals in any vertebrate animal model.