Chlorophyllin significantly reduces benzo[a]pyrene-DNA adduct formation and alters cytochrome P450 1A1 and 1B1 expression and EROD activity in normal human mammary epithelial cells.
We hypothesized that chlorophyllin (CHLN) would reduce benzo[a]pyrene-DNA (BP-DNA) adduct levels. Using normal human mammary epithelial cells (NHMECs) exposed to 4 microM BP for 24 hr in the presence or absence of 5 microM CHLN, we measured BP-DNA adducts by chemiluminescence immunoassay (CIA). The protocol included the following experimental groups: BP alone, BP given simultaneously with CHLN (BP+CHLN) for 24 hr, CHLN given for 24 hr followed by BP for 24 hr (preCHLN, postBP), and CHLN given for 48 hr with BP added for the last 24 hr (preCHLN, postBP+CHLN). Incubation with CHLN decreased BPdG levels in all groups, with 87% inhibition in the preCHLN, postBP+CHLN group. To examine metabolic mechanisms, we monitored expression by Affymetrix microarray (U133A), and found BP-induced up-regulation of CYP1A1 and CYP1B1 expression, as well as up-regulation of groups of interferon-inducible, inflammation and signal transduction genes. Incubation of cells with CHLN and BP in any combination decreased expression of many of these genes. Using reverse transcription real time PCR (RT-PCR) the maximal inhibition of BP-induced gene expression, >85% for CYP1A1 and >70% for CYP1B1, was observed in the preCHLN, postBP+CHLN group. To explore the relationship between transcription and enzyme activity, the ethoxyresorufin-O-deethylase (EROD) assay was used to measure the combined CYP1A1 and CYP1B1 activities. BP exposure caused the EROD levels to double, when compared with the unexposed controls. The CHLN-exposed groups all showed EROD levels similar to the unexposed controls. Therefore, the addition of CHLN to BP-exposed cells reduced BPdG formation and CYP1A1 and CYP1B1 expression, but EROD activity was not significantly reduced.
Keshava
Channa
C
Toxicology and Molecular Biology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia, USA.
Divi
Rao L
RL
Einem
Tracey L
TL
Richardson
Diana L
DL
Leonard
Sarah L
SL
Keshava
Nagalakshmi
N
Poirier
Miriam C
MC
Weston
Ainsley
A
D016587:Antimutagenic Agents / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases; D001564:Benzo(a)pyrene / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002735:Chlorophyllides / Q000494:pharmacology*; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics* / Q000378:metabolism; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D018736:DNA Adducts / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling; D006801:Humans; D008163:Luminescent Measurements; D042361:Mammary Glands, Human / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016587:Antimutagenic Agents; D002735:Chlorophyllides; D018736:DNA Adducts; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1; C007020:chlorophyllin