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

Abstract

We hypothesized that chlorophyllin (CHLN) would reduce BP-DNA adduct levels. Using NHMECs exposed to 4 μM BP for 24 hr in the presence or absence of 5 μM 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 interferoninducible, inflammation and signal transduction genes. Incubation of cells with CHLN and BP in any combination decreased expression of many of these genes. Using 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, compared to 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.

CYP1A1 and CYP1B1 gene expression and DNA adduct formation in normal human mammary epithelial cells exposed to benzo[a]pyrene in the absence or presence of chlorophyllin

“…The gene expression experiments presented here expand our previous work using DNA microarrays in a single NHMEC strain, M98013 [16]. M98013 was used in this study and values for BP-induced CYP1A1 and CYP1B1 expression were similar to those found previously [16], however, the addition of 19 new NHMEC strains revealed considerable variation in levels of BP-induced CYP1A1 and CYP1B1 expression.…”

“…M98013 was used in this study and values for BP-induced CYP1A1 and CYP1B1 expression were similar to those found previously [16], however, the addition of 19 new NHMEC strains revealed considerable variation in levels of BP-induced CYP1A1 and CYP1B1 expression. This wide inter-individual variation might be explained, at least in part by polymorphisms in the genes coding for cytochrome P450s and other Phase I enzymes, as well as genes coding for Phase II or detoxication enzymes and DNA repair functions [11].…”

Copper chlorophyllin: A food colorant with bioactive properties?

“…Abraham et al (1994) suggested that Cu-Chl absorbed in the gastrointestinal tract may inhibit the activation process of clastogens by modulating the activity of enzymes, such as cytochrome P450. In studies using NHMECs, Keshava et al (2009) observed that treatment with Cu-Chl reduced the expression of several genes, including some that encode cytochrome P450 enzymes. Other studies also suggest that chlorophyllin may bind to RNA (Marty, Ouameur, Neault, & Tajmir-Riahi, 2004), which results in inhibition of translation, or intercalate with DNA (Tajmir-Riahi, Neault, & Diamantoglou, 2004) by binding with cytochrome P450 genes.…”

“…Down-regulation of gene expression in normal NHMECs (strain 98013) exposed to BP (4 μM) 5 μM (cells exposed for 24 h, washed and exposed to BP+ Chl for 24 h) Cu-Chl, Sigma Aldrich Keshava et al, 2009 Cu-Chl displayed maximum inhibition of BP-induced gene expression (>85% for CYP1A1 gene and >70% for CYP1B1), and down-regulated other genes (interferon-associated, inflammatory, signal transduction and metabolism genes).…”

Transcriptional toxicity of the Yangtze River source water on mouse (Mus musculus) detected by cDNA microarray

“…The DEGs involved in this metabolic process of mouse liver mainly included the aberrant regulation of Cyps and Gsts. It has been proved that many xenobiotics such as toxins and environmental pollutants could induce the aberrant expression of Cyps and Gsts (Anderson et al 1998;Crosby et al 2008;Keshava et al 2009). For example, Mei et al(2008) found that acrylamide could significantly affect the xenobiotic metabolism by cytochrome P450 and glutathione metabolism in the mouse liver.…”