Abstract
In the past, CYP1A1 protein was known to be located in the endoplasmic reticulum (ER; microsomes). More recently, CYP1A1 was shown also to be targeted to the inner mitochondrial membrane; mitochondrial import is dependent on NH2-terminal processing that exposes a cryptic targeting signal. It is interesting that microsomal and mitochondrial CYP1A1 enzymes exhibit different substrate specificities, electron donors, and inducer properties. To understand the physiological functions of microsomal versus mitochondrial CYP1A1, we have generated three knock-in lines by altering the CYP1A1 NH2 terminus. Cyp1a1(mtt/mtt) mice encode an NH2-terminal 31-amino acid-truncated protein, deleting the ER-targeting signal and exposing the cryptic mitochondrial-targeting signal. Cyp1a1(mtp/mtp) mice encode a protein carrying L7N and L17N mutations; this mutant lacks the signal recognition particle (SRP)-binding site and subsequent ER-targeting, but requires proteolysis by a cytosolic peptidase for mitochondrial import. Cyp1a1(mc/mc) mice encode a microsomal protein having R34D and K39I mutations, which abolish the mitochondrial targeting signal. After dioxin or β-naphthoflavone treatment of these mouse lines, the CYP1A1 protein was shown to be located in the mitochondria of the Cyp1a1(mtp/mtp) and Cyp1a1(mtt/mtt) lines and in microsomes of the Cyp1a1(mc/mc) line. To test for differences in function, we compared the response to dietary benzo[a]pyrene (BaP). After 18 days of daily oral BaP, wild-type and Cyp1a1(mc/mc) mice were completely protected, whereas Cyp1a1(-/-) and Cyp1a1(mtp/mtp) mice showed striking toxicity and compensatory up-regulation of CYP1A2 and CYP1B1 mRNA in several tissues. Our data support the likelihood that it is the microsomal rather than mitochondrial CYP1A1 enzyme that protects against oral BaP toxicity.
Footnotes
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These studies were supported, in part, by the National Institutes of Health National Institute of Environmental Health Sciences [Grants R01-ES08147, R01-ES014403, P30-ES06096] and the National Institutes of Health National Institute of General Medical Sciences [Grant R01-GM034883].
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These data were presented in part at the 26th (March 5-9, 2006; San Diego, CA) and 27th (March 25-29, 2007; Charlotte, NC) Annual Meetings of the Society of Toxicology.
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ABBREVIATIONS: AHR, aryl hydrocarbon receptor; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BaP, benzo[a]pyrene; BNF, β-naphthoflavone; ER, endoplasmic reticulum; MT, mitochondrial; POR, NADPH-P450 oxidoreductase; PHB, prohibitin; GCLM, glutamate-cysteine ligase modifier subunit; SRP, signal-recognition particle; ERND, erythromycin N-demethylase; FDX1, ferredoxin-1; FDXR, ferredoxin reductase; CYP1A1, full-length translated protein from the mouse Cyp1a1 gene; mc1A1, microsomal (endoplasmic reticulum)-targeted CYP1A1 protein; mt1A1, mitochondrial-targeted CYP1A1 protein; Cyp1a1(+/+) or WT 1A1, wild-type (C57BL/6J) mouse; Cyp1a1(-/-) or KO 1A1, mouse line having global knockout of the Cyp1a1 gene; Cyp1a1(mc/mc) or mc1A1, line carrying endoplasmic reticulum-targeted CYP1A1 protein; Cyp1a1(mtp/mtp) or mtp1A1, line carrying mitochondrial-targeted CYP1A1 protein via proteolysis; Cyp1a1(mtt/mtt) or mtt1A1, line carrying mitochondrial-targeted CYP1A1 protein via truncation; TCDD, dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin; PAGE, polyacrylamide gel electrophoresis; PKC, protein kinase C; KO, knockout; bp, base pair(s); kb, kilobase pair(s); TK, thymidine kinase; ES, embryonic stem; PCR, polymerase chain reaction; SSC, standard saline citrate; WT, wild type; qRT-PCR, quantitative real-time polymerase-chain reaction; SKF-525A, 2-diethyl-aminoethyl-2,2-diphenylvalerate hydrochloride; f, forward; r, reverse.
- Received September 8, 2008.
- Accepted December 1, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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Knock-In Mouse Lines Expressing either Mitochondrial or Microsomal CYP1A1: Differing Responses to Dietary Benzo[a]pyrene as Proof of Principle
