Identifying efficacious approaches to chemoprevention with chlorophyllin, purified chlorophylls and freeze-dried spinach in a mouse model of transplacental carcinogenesis

Abstract

The carcinogenic potential of dibenzo[a,l]pyrene (DBP) has been well characterized in numerous animal models. We have previously documented that a single dose of 15 mg/Kg DBP to pregnant mice late in gestation (GD 17) produces an aggressive T-cell lymphoma as well as lung and liver cancer in offspring. The current study examines the chemopreventative properties of chlorophyllin (CHL) and chlorophyll (Chl) in this transplacental carcinogenesis model. Pregnant B6129SF1 females, bred to 129S1/SvIm males, received purified diets incorporated with either 2000 p.p.m. CHL, 2000 p.p.m. Chl or 10% freeze-dried spinach beginning at gestation day 9. Lymphoma-dependent mortality was not significantly altered by maternal consumption of any of the diet and little effect on lung tumor burden in mice surviving to 10 months of age was observed. However, coadministration of CHL at 380 mg/Kg with DBP by gavage (molar ratio of 10:1, CHL:DBP) provided significant protection against DBP-initiated carcinogenesis. Offspring born to dams receiving CHL co-gavaged with DBP exhibited markedly less lymphoma-dependent mortality (P < 0.001). The degree of protection by CHL, compared with controls dosed with DBP in tricaprylin (TCP) as the vehicle, was less marked, but still significant. Coadministration of CHL (TCP as vehicle) also reduced lung tumor multiplicity in mice by approximately 50% and this was observed throughout the study (P < 0.005). This is the first demonstration that CHL can provide potent chemoprotection in a transplacental carcinogenesis model and support a mechanism involving complex-mediated reduction of carcinogen uptake.

Fig. 1.

Effect of maternal dietary treatment on DBP-dependent mortality. DBP was administered at a dose of 15 mg/Kg by gavage (corn oil) on day 17 of gestation. The pregnant and nursing dams were administered powdered AIN93G diets supplemented with nothing [control (open squares)], 2000 p.p.m. CHL (filled triangles), 2000 p.p.m. Chl (inverted triangle-dashed line) or 10% freeze-dried spinach (inverted triangle-solid line).

Fig. 2.

(A) Impact of coadministration of CHL on DBP-dependent mortality. Pregnant and nursing dams were administered control diets and gavaged at gestation day 17 with DBP in corn oil (filled triangles), TCP (filled inverted triangles) or DBP concurrently with 380 mg/kg CHL (open squares). CHL was coadministered at a dose of 380 mg/kg by co-gavage with DBP (15 mg/kg) to give a molar ratio of 10 (CHL:DBP). There is strong statistical evidence of vehicle differences (P < 0.001 for DBP in corn oil versus co-CHL and P < 0.01 for DBP in TCP versus co-CHL. Likewise, DBP in corn oil versus TCP was significant, P < 0.01) (B) Influence of genotype and CHL co-gavage on DBP-dependent mortality. Offspring born to dams receiving DBP by gavage (TCP as the vehicle) and genotyped as ahr^b-1/d (open diamonds) or ahr^d/d (filled diamonds); offspring born to dams receiving DBP concurrently with 380 mg/kg CHL (in TCP) and genotyped as ahr^b-1/d (open squares) or ahr^d/d (filled squares). P-values associated with treatment differences indicate significance regardless of genotype upon co-CHL administration (P < 0.01).

Fig. 3.

Effects of coadministered CHL on lung tumor burden throughout the study. Offspring were euthanized due to lymphoma-dependent morbidity (3–9 months) or at the conclusion of the study (10 months) and lung lesions (predominantly adenomas) quantified by histopathology. Data indicate offspring born to mothers given 15 mg/kg DBP alone by gavage (in TCP) (open squares) or concurrent with 380 mg/kg CHL (in TCP) (inverted filled triangles). Numbers euthanized in the first group (<21 weeks) were 7 and 2. For the second group (>21 weeks and <44 weeks), 8 and 16 were euthanized. Numbers surviving to the end were 30 and 82 for control-B and co-CHL, respectively. The bars indicate ± SE.