Biotin supplementation increases expression of the cytochrome P450 1B1 gene in Jurkat cells, increasing the occurrence of single-stranded DNA breaks.
DNA microarray studies provided evidence that biotin supplementation increases the abundance of mRNA encoding cytochrome P(450) 1B1 (CYP1B1) in human lymphocytes. CYP1B1 hydroxylates procarcinogens, generating electrophilic mutagens. Here, we sought to identify the signaling pathways that increase the expression of CYP1B1 in biotin-supplemented human T (Jurkat) cells and to determine whether activation of the CYP1B1 gene is associated with increased occurrence of single-stranded DNA breaks. Jurkat cells were cultured in biotin-deficient (0.025 nmol/L) and biotin-supplemented (10 nmol/L) media. The transcriptional activity of a CYP1B1 reporter gene construct was 24% greater in biotin-supplemented compared with biotin-deficient cells (P < 0.01). Similarly, the abundance of CYP1B1 mRNA was 72% greater in biotin-supplemented than in biotin-deficient cells (P < 0.05). Electrophoretic mobility shift assays suggested that Sp1 sites in the regulatory region of the CYP1B1 gene play important roles in transcriptional activation by biotin. The abundance of CYP1B1 protein and activity of CYP1B1 were 124 and 35% greater, respectively, in biotin-supplemented compared with biotin-deficient cells (P < 0.05). The increased expression of CYP1B1 in biotin-supplemented cells was associated with an increase in the occurrence of single-stranded DNA breaks compared with biotin-deficient cells; synthetic inhibitors of CYP1B1 prevented strand breaks, suggesting that the effects of biotin were specific for CYP1B1. These studies provide evidence that transcription factors with an affinity for Sp1 sites mediate transcriptional activation of the CYP1B1 gene in biotin-supplemented T cells, increasing the occurrence of single-stranded DNA breaks.
Rodriguez-Melendez
Rocio
R
Departments of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE, USA.
Griffin
Jacob B
JB
Zempleni
Janos
J
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D001710:Biotin / Q000378:metabolism / Q000494:pharmacology*; D015153:Blotting, Western; D002262:Carboxy-Lyases / Q000378:metabolism; D065633:Cytochrome P-450 CYP1B1; D004249:DNA Damage / Q000187:drug effects*; D004277:DNA, Single-Stranded; D004586:Electrophoresis; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D019169:Jurkat Cells; D008025:Ligases / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic
D004277:DNA, Single-Stranded; D001710:Biotin; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1; D002262:Carboxy-Lyases; D008025:Ligases