Chlorophyllin intervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer

Abstract

Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part from consumption of foods contaminated with aflatoxins. Chlorophyllin, a mixture of semisynthetic, water-soluble derivatives of chlorophyll that is used as a food colorant and over-the-counter medicine, has been shown to be an effective inhibitor of aflatoxin hepatocarcinogenesis in animal models by blocking carcinogen bioavailability. In a randomized, double-blind, placebo-controlled chemoprevention trial, we tested whether chlorophyllin could alter the disposition of aflatoxin. One hundred and eighty healthy adults from Qidong were randomly assigned to ingest 100 mg of chlorophyllin or a placebo three times a day for 4 months. The primary endpoint was modulation of levels of aflatoxin-N(7)-guanine adducts in urine samples collected 3 months into the intervention measured by using sequential immunoaffinity chromatography and liquid chromatography-electrospray mass spectrometry. This aflatoxin-DNA adduct excretion product serves as a biomarker of the biologically effective dose of aflatoxin, and elevated levels are associated with increased risk of liver cancer. Adherence to the study protocol was outstanding, and no adverse events were reported. Aflatoxin-N(7)-guanine could be detected in 105 of 169 available samples. Chlorophyllin consumption at each meal led to an overall 55% reduction (P = 0.036) in median urinary levels of this aflatoxin biomarker compared with those taking placebo. Thus, prophylactic interventions with chlorophyllin or supplementation of diets with foods rich in chlorophylls may represent practical means to prevent the development of hepatocellular carcinoma or other environmentally induced cancers.

Figures

Figure 1

Pathway for formation of aflatoxin-N7-guanine after exposure to aflatoxin B1.

Figure 2

HPLC tracing (A626) of the copper chlorin constituents of the Derifil used in the intervention. Analysis was conducted as described in ref. .

Figure 3

Chlorophyllin chemoprevention trial profile.

Figure 4

Liquid chromatography–electrospray mass spectrometry of aflatoxin-N7-guanine isolated from the urine (0.32 pg/mg creatinine) of a study participant. (A) Chromatogram of the HPLC separation of aflatoxin-N7-guanine after isolation from urine by immunoaffinity chromatography. The profile illustrates the elution of the daughter ion 152.1 MH+ formed from the fragmentation of the parent compound, aflatoxin-N7-guanine (m/z 480.1) at 10.9 min. (B) Two major fragmentation ions obtained from urinary aflatoxin-N7-guanine. (C) HPLC elution of the daughter ion 259.1 MH+ fragmented from the spiked internal standard, aflatoxin B2 (m/z 315.1), which elutes at 20.6 min. (D) Fragmentation pattern for aflatoxin B2.

Figure 5

Geometric means and 95% confidence intervals for the distribution of detectable values for excreted amounts of aflatoxin-N7-guanine 12 weeks after thrice-daily administration of placebo or chlorophyllin (CHL, 100 mg) tablets. RL, relative level; NS, not significant.