Knockout of cytochrome P450 1A1 enhances lipopolysaccharide-induced acute lung injury in mice by targeting NF-κB activation.

Acute lung injury (ALI) is accompanied by overactivation of multiple pro-inflammatory factors. Cytochrome P450 1A1 (CYP1A1) has been shown to aggravate lung injury in response to hyperoxia. However, the relationship between CYP1A1 and lipopolysaccharide (LPS)-induced ALI is unknown. In this study, CYP1A1 was shown to be upregulated in mouse lung in response to LPS. Using CYP1A1-deficient (CYP1A1-/-) mice, we found that CYP1A1 knockout enhanced LPS-induced ALI, as evidenced by increased TNF-α, IL-1ß, IL-6, and nitric oxide in lung; these effects were mediated by overactivation of NF-κB and iNOS. Furthermore, we found that aspartate aminotransferase, lactate dehydrogenase, creatine kinase, and creatinine levels were elevated in serum of LPS-induced CYP1A1-/- mice. Altogether, these data provide novel insights into the involvement of CYP1A1 in LPS-induced lung injury.

Differential effects of variations in human P450 oxidoreductase on the aromatase activity of CYP19A1 polymorphisms R264C and R264H.

Aromatase (CYP19A1) converts androgens into estrogens and is required for female sexual development and growth and development in both sexes. CYP19A1 is a member of cytochrome P450 family of heme-thiolate monooxygenases located in the endoplasmic reticulum and depends on reducing equivalents from the reduced nicotinamide adenine dinucleotide phosphate via the cytochrome P450 oxidoreductase coded by POR. Both the CYP19A1 and POR genes are highly polymorphic, and mutations in both these genes are linked to disorders of steroid biosynthesis. We have previously shown that R264C and R264H mutations in CYP19A1, as well as mutations in POR, reduce CYP19A1 activity. The R264C is a common polymorphic variant of CYP19A1, with high frequency in Asian and African populations. Polymorphic alleles of POR are found in all populations studied so far and, therefore, may influence activities of CYP19A1 allelic variants. So far, the effects of variations in POR on enzymatic activities of allelic variants of CYP19A1 or any other steroid metabolizing cytochrome P450 proteins have not been studied. Here we are reporting the effects of three POR variants on the aromatase activities of two CYP19A1 variants, R264C, and R264H. We used bacterially expressed and purified preparations of WT and variant forms of CYP19A1 and POR and constructed liposomes with embedded CYP19A1 and POR proteins and assayed the CYP19A1 activities using radiolabeled androstenedione as a substrate. With the WT-POR as a redox partner, the R264C-CYP19A1 showed only 15% of aromatase activity, but the R264H had 87% of aromatase activity compared to WT-CYP19A1. With P284L-POR as a redox partner, R264C-CYP19A1 lost all activity but retained 6.7% of activity when P284T-POR was used as a redox partner. The R264H-CYP19A1 showed low activities with both the POR-P284 L as well as the POR-P284 T. When the POR-Y607C was used as a redox partner, the R264C-CYP19A1 retained approximately 5% of CYP19A1 activity. Remarkably, The R264H-CYP19A1 had more than three-fold higher activity compared to WT-CYP19A1 when the POR-Y607C was used as the redox partner, pointing toward a beneficial effect. The slight increase in activity of R264C-CYP19A1 with the P284T-POR and the three-fold increase in activity of the R264H-CYP19A1 with the Y607C-POR point toward a conformational effect and role of protein-protein interaction governed by the R264C and R264H substitutions in the CYP19A1 as well as P284 L, P284 T and Y607C variants of POR. These studies demonstrate that the allelic variants of P450 when present with a variant form of POR may show different activities, and combined effects of variations in the P450 enzymes as well as in the POR should be considered when genetic data are available. Recent trends in the whole-exome and whole-genome sequencing as diagnostic tools will permit combined evaluation of variations in multiple genes that are interdependent and may guide treatment options by adjusting therapeutic interventions based on laboratory analysis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Induces Vascular Dysfunction That is Dependent on Perivascular Adipose and Cytochrome P4501A1 Expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is associated with hypertension in humans and animals, and studies suggest that cytochrome P4501A1 (Cyp1a1) induction and vascular dysfunction may contribute. We investigated the role of perivascular adipose tissue (PVAT) and Cyp1a1 in TCDD-induced vascular dysfunction. Cyp1a1 wild-type (WT) and knockout (KO) male mice were fed a dough pill containing 1,4-p-dioxane (TCDD vehicle control) on days 0 and 7, or 1000 ng/kg TCDD on day 0 and 250 ng/kg TCDD on day 7. mRNA expression of Cyp1a1 was assessed on days 3, 7, and 14, and of Cyp1b1, 1a2, angiotensinogen, and phosphodiesterase 5a on day 14. Dose-dependent vasoconstriction to a thromboxane A2 mimetic (U46619), and vasorelaxation to acetylcholine and a nitric oxide donor (S-nitroso-N-acetyl-DL-penicillamine, SNAP), were investigated in the aorta with and without PVAT. Cyp1a1 and 1a2 mRNA was induced in aorta of WT mice only with PVAT, and Cyp1a1 induction was sustained through day 14. TCDD significantly enhanced constriction to U46619 in WT mice and inhibited relaxation to both acetylcholine and SNAP, but only in the presence of PVAT. The effects of TCDD on U46619 constriction and SNAP relaxation were not observed in Cyp1a1 KO mice. Finally, in aorta + PVAT of WT mice TCDD significantly induced expression of angiotensinogen and phosphodiesterase 5a both of which could contribute to the TCDD-induced vascular dysfunction. These data establish PVAT as a TCDD target which is critically involved in mediating vascular dysfunction. TCDD enhances vasoconstriction via the thromboxane/prostanoid (TP) receptor and inhibits vasorelaxation via nitric oxide (NO) signaling. This TCDD-induced vascular dysfunction requires perivascular adipose (PVAT) and cytochrome P4501a1 (CYP1a1) induction.

Biological effects of 6-formylindolo[3,2-b]carbazole (FICZ) in vivo are enhanced by loss of CYP1A function in an Ahr2-dependent manner.

6-Formylindolo[3,2-b]carbazole (FICZ) is a potent aryl hydrocarbon receptor (AHR) agonist that is efficiently metabolized by AHR-regulated cytochrome P4501 enzymes. FICZ is a proposed physiological AHR ligand that induces its own degradation as part of a regulatory negative feedback loop. In vitro studies in cells show that CYP1 inhibition in the presence of FICZ results in enhanced AHR activation, suggesting that FICZ accumulates in the cell when its metabolism is blocked. We used zebrafish (Danio rerio) embryos to investigate the in vivo effects of FICZ when CYP1A is knocked down or inhibited. Embryos were injected with morpholino antisense oligonucleotides targeting CYP1A (CYP1A-MO), Ahr2, or a combination of both. FICZ exposure of non-injected embryos or embryos injected with control morpholino had little effect. In CYP1A-MO-injected embryos, however, FICZ dramatically increased mortality, incidence and severity of pericardial edema and circulation failure, reduced hatching frequency, blocked swim bladder inflation, and strongly potentiated expression of Ahr2-regulated genes. These effects were substantially reduced in embryos with a combined knockdown of Ahr2 and CYP1A, indicating that the toxicity was mediated at least partly by Ahr2. Co-exposure to the CYP1 inhibitor alpha-naphthoflavone (αNF) and FICZ had similar effects as the combination of CYP1A-MO and FICZ. HPLC analysis of FICZ-exposed embryos showed increased levels of FICZ after concomitant CYP1A-MO injection or αNF co-exposure. Together, these results show that a functioning CYP1/AHR feedback loop is crucial for regulation of AHR signaling by a potential physiological ligand in vivo and further highlights the role of CYP1 enzymes in regulating biological effects of FICZ.

Sex-specific differences in hyperoxic lung injury in mice: role of cytochrome P450 (CYP)1A.

Sex-specific differences in pulmonary morbidity in adults and preterm infants are well documented. Hyperoxia contributes to lung injury in experimental animals and humans. Cytochrome P450 (CYP) 1A enzymes have been shown to play a mechanistic role in hyperoxic lung injury (HLI) in animal models. Whether CYP1A enzymes contribute to gender-specific differences in relation to HLI is unknown. In this investigation, we tested the hypothesis that mice will display gender-specific differences in HLI, and that this phenomenon will be altered in mice lacking the genes for Cyp1a1 or 1a2. Eight week-old male and female wild type (WT) (C57BL/6J) mice, Cyp1a1-/-, and Cyp1a2-/- mice were exposed to 72h of hyperoxia (FiO2>0.95). Lung injury and inflammation were assessed and pulmonary and hepatic CYP1A1 and CYP1A2 levels were quantified at the enzyme activity, protein and mRNA level. Upon exposure to hyperoxia, liver and lung microsomal proteins showed higher pulmonary CYP1A1 (apoprotein level and activity) in WT females compared to WT males and a greater induction in hepatic CYP1A2 mRNA levels and activity in WT females after hyperoxia exposure. The gender based female advantage was lost or reversed in Cyp1a1-/- and Cyp1a2-/- mice. These findings suggest an important role for CYP1A enzymes in the gender-specific modulation of hyperoxic lung injury.

The anticancer drug ellipticine activated with cytochrome P450 mediates DNA damage determining its pharmacological efficiencies: studies with rats, Hepatic Cytochrome P450 Reductase Null (HRN™) mice and pure enzymes.

Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b5 alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b5, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models.

Smoke carcinogens cause bone loss through the aryl hydrocarbon receptor and induction of Cyp1 enzymes.

Smoking is a major risk factor for osteoporosis and fracture, but the mechanism through which smoke causes bone loss remains unclear. Here, we show that the smoke toxins benzo(a)pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) interact with the aryl hydrocarbon receptor (Ahr) to induce osteoclastic bone resorption through the activation of cytochrome P450 1a/1b (Cyp1) enzymes. BaP and TCDD enhanced osteoclast formation in bone marrow cell cultures and gavage with BaP stimulated bone resorption and osteoclastogenesis in vivo. The osteoclastogenesis triggered by BaP or RANK-L was reduced in Ahr(-/-) cells, consistent with the high bone mass noted in Ahr(-/-) male mice. The receptor activator of NF-κB ligand (RANK-L) also failed to induce the expression of Cyp1 enzymes in Ahr(-/-) cells. Furthermore, the osteoclastogenesis induced by TCDD was lower in Cyp1a1/1a2(-/-) and Cyp1a1/1a2/1b1(-/-) cultures, indicating that Ahr was upstream of the Cyp enzymes. Likewise, the pharmacological inhibition of the Cyp1 enzymes with tetramethylsilane or proadifen reduced osteoclastogenesis. Finally, deletion of the Cyp1a1, Cyp1a2, and Cyp1b1 in triple knockout mice resulted in reduced bone resorption and recapitulated the high bone mass phenotype of Ahr(-/-) mice. Overall, the data identify the Ahr and Cyp1 enzymes not only in the pathophysiology of smoke-induced osteoporosis, but also as potential targets for selective modulation by new therapeutics.

NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I.

Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)-the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1⁻/⁻, Cyp1a2⁻/⁻ and Cyp1a1/1a2⁻/⁻ knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.

Role of P450 1A1 and P450 1A2 in bioactivation versus detoxication of the renal carcinogen aristolochic acid I: studies in Cyp1a1-/-, Cyp1a2-/-, and Cyp1a1/1a2-/- mice.

Exposure to aristolochic acid I (AAI) is associated with aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial cancer. Individual differences in xenobiotic-metabolizing enzyme activities are likely to be a reason for interindividual susceptibility to AA-induced disease. We evaluated the reductive activation and oxidative detoxication of AAI by cytochrome P450 (P450) 1A1 and 1A2 using the Cyp1a1(-/-) and Cyp1a2(-/-) single-knockout and Cyp1a1/1a2(-/-) double-knockout mouse lines. Incubations with hepatic microsomes were also carried out in vitro. P450 1A1 and 1A2 were found to (i) activate AAI to form DNA adducts and (ii) detoxicate it to 8-hydroxyaristolochic acid I (AAIa). AAI-DNA adduct formation was significantly higher in all tissues of Cyp1a1/1a2(-/-) than Cyp1a(+/+) wild-type (WT) mice. AAI-DNA adduct levels were elevated only in selected tissues from Cyp1a1(-/-) versus Cyp1a2(-/-) mice, compared with those in WT mice. In hepatic microsomes, those from WT as well as Cyp1a1(-/-) and Cyp1a2(-/-) mice were able to detoxicate AAI to AAIa, whereas Cyp1a1/1a2(-/-) microsomes were less effective in catalyzing this reaction, confirming that both mouse P450 1A1 and 1A2 are both involved in AAI detoxication. Under hypoxic conditions, mouse P450 1A1 and 1A2 were capable of reducing AAI to form DNA adducts in hepatic microsomes; the major roles of P450 1A1 and 1A2 in AAI-DNA adduct formation were further confirmed using selective inhibitors. Our results suggest that, in addition to P450 1A1 and 1A2 expression levels in liver, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.

Generation of 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-) mouse line.

Human/rodent CYP1A1 and CYP1A2 orthologs are well known to exhibit species-specific differences in substrate preferences and rates of metabolism. This lab previously characterized a BAC-transgenic mouse carrying the human CYP1A1_CYP1A2 locus; in this line, human dioxin-inducible CYP1A1 and basal vs dioxin-inducible CYP1A2 have been shown to be expressed normally (with regard to mRNAs, proteins and three enzyme activities) in every one of nine mouse tissues studied. The mouse Cyp1a1 and Cyp1a2 genes are oriented head-to-head and share a bidirectional promoter region of 13,954 bp. Using Cre recombinase and loxP sites inserted 3' of the stop codons of both genes, we show here a successful interchromosomal excision of 26,173 bp that ablated both genes on the same allele. The Cyp1a1/1a2(-) double-knockout allele was bred with the "humanized" line; the final product is the hCYP1A1_1A2_Cyp1a1/1a2(-/-) line on a theoretically >99.8% C57BL/6J genetic background-having both human genes replacing the mouse orthologs. This line will be valuable for human risk assessment studies involving any environmental toxicant or drug that is a substrate for CYP1A1 or CYP1A2.

Mouse lung CYP1A1 catalyzes the metabolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis is initiated by N(2)-hydroxylation, mediated by several cytochromes P450, including CYP1A1. However, the role of CYP1A1 in PhIP metabolic activation in vivo is unclear. In this study, Cyp1a1-null and wild-type (WT) mice were used to investigate the potential role of CYP1A1 in PhIP metabolic activation in vivo. PhIP N(2)-hydroxylation was actively catalyzed by lung homogenates of WT mice, at a rate of 14.9 +/- 5.0 pmol/min/g tissue, but <1 pmol/min/g tissue in stomach and small intestine, and almost undetectable in mammary gland and colon. PhIP N(2)-hydroxylation catalyzed by lung homogenates of Cyp1a1-null mice was approximately 10-fold lower than that of WT mice. In contrast, PhIP N(2)-hydroxylation activity in lung homogenates of Cyp1a2-null versus WT mice was not decreased. Pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin increased lung Cyp1a1 mRNA and lung homogenate PhIP N(2)-hydroxylase activity approximately 50-fold in WT mice, where the activity was substantially inhibited (70%) by monoclonal antibodies against CYP1A1. In vivo, 30 min after oral treatment with PhIP, PhIP levels in lung were similar to those in liver. After a single dose of 0.1 mg/kg [(14)C]PhIP, lung PhIP-DNA adduct levels in Cyp1a1-null mice, but not in Cyp1a2-null mice, were significantly lower (P = 0.0028) than in WT mice. These results reveal that mouse lung has basal and inducible PhIP N(2)-hydroxylase activity predominantly catalyzed by CYP1A1. Because of the high inducibility of human CYP1A1, especially in cigarette smokers, the role of lung CYP1A1 in PhIP carcinogenesis should be considered. (237 words).

AT1 receptor blockade is superior to conventional triple therapy in protecting against end-organ damage in Cyp1a1-Ren-2 transgenic rats with inducible hypertension.

OBJECTIVE: In the present study we compared the effects of treatment with the AT1 receptor antagonist candesartan and of 'triple therapy' (hydralazine, hydrochlorothiazide, reserpine) on the course of blood pressure, cardiac hypertrophy and angiotensin II concentrations after induction of hypertension in transgenic rats with inducible expression of the mouse renin gene (Cyp1a1-Ren-2 rats). METHODS: Hypertension was induced in Cyp1a1-Ren-2 rats through dietary administration of the natural xenobiotic indole-3-carbinol (I3C, 0.3%) for 4 days. Starting on the day before administration of I3C, rats were treated either with candesartan or received triple therapy for 9 days. Systolic blood pressure was measured in conscious animals. Rats were decapitated and angiotensin II levels in plasma and in whole kidney and left ventricular tissues were determined by radioimmunoassay. RESULTS: Administration of I3C resulted in the development of severe hypertension and cardiac hypertrophy that was accompanied by marked elevations of plasma and tissue angiotensin II concentrations. Candesartan treatment prevented the development of hypertension and cardiac hypertrophy and was associated with a reduction of tissue angiotensin II concentrations. In contrast, triple therapy, despite maintaining systolic blood pressure in the normotensive range, did not prevent the development of cardiac hypertrophy and tissue angiotensin II augmentations. CONCLUSIONS: Our findings indicate that hypertension in Cyp1a1-Ren-2 rats is a clearly angiotensin II-dependent model of hypertension with elevated circulating and tissue angiotensin II concentrations, and that antihypertensive treatment with AT1 receptor blockade is superior to conventional triple therapy in effective protection against hypertension-induced end-organ damage in this rat model.

Lack of CYP1A1 expression is involved in unresponsiveness of the human hepatoma cell line SK-HEP-1 to dioxin.

The aryl hydrocarbon receptor (AhR) mediates a wide variety of toxic effects due to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The human hepatoma cell line SK-HEP-1 expresses AhR and ARNT. However, TCDD failed to induce CYP1A1 and XRE-dependent reporter genes in these cells. Although CYP1A1 was not induced by TCDD exposure, both CYP1B1 and AhR repressor (AhRR) were constitutively expressed. The AhR antagonist alpha-naphthoflavone altered the basal level of XRE-dependent reporter gene expression dose-dependently. As our results suggested the activation of AhR signals by putative endogenous ligands, we established SK-HEP-1-derived cell lines that stably expressed CYP1A1. The inducibility of XRE-dependent reporter genes and CYP1B1 by TCDD was restored in these cells. Our findings demonstrated the presence of endogenous ligands in SK-HEP-1 cells due to the absence of the metabolizing enzyme CYP1A1, but not CYP1B1, which allowed the constitutive expression of AhR target genes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin activation of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator pathway causes developmental toxicity through a CYP1A-independent mechanism in zebrafish.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that dimerizes with ARNT to mediate responses to compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD and other AHR agonists cause toxic responses in early life stages of fish, including the zebrafish, Danio rerio. The most well characterized target gene for the AHR/aryl hydrocarbon receptor nuclear translocator (ARNT) dimer is a cytochrome P450, CYP1A. Induction of CYP1A by TCDD has been correlated with certain toxic responses in developing zebrafish and has been postulated to mediate these responses. To determine whether CYP1A is the important downstream effector enzyme for the AHR/ARNT pathway, we used morpholino oligonucleotides (MOs) to block induction of CYP1A in response to TCDD in zebrafish embryos. Although the zfcyp1a-MO effectively prevented CYP1A up-regulation, it did not prevent the signs of developmental toxicity, including pericardial edema, slowed blood flow, craniofacial malformation, and defects in erythropoiesis. We conclude that the important target for the AHR/ARNT pathway in developing zebrafish exposed to TCDD is not zfcyp1a. This suggests an alternative model in which TCDD-activated AHR/ARNT disrupts the normal process of growth and development by altering programs of gene expression or function.

Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1(-/-) knockout mice having increased hepatic BaP-DNA adduct levels.

Previous studies have shown that cytochrome P450 1A1 (CYP1A1), CYP1B1, and prostaglandin-endoperoxide synthase (PTGS2) are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and all three metabolize BaP to reactive DNA-binding intermediates and excreted products. Because these three enzymes show differing patterns of basal levels, inducibility, and tissue-specific expression, animal studies are necessary to delineate the role of CYP1A1 in BaP-mediated toxicity. In mice receiving large daily doses of BaP (500 mg/kg i.p.), Cyp1a1(-/-) knockout mice are protected by surviving longer than Cyp1a1(+/-) heterozygotes. We found that a single 500 mg/kg dose of BaP induces hepatic CYP1A1 mRNA, protein, and enzyme activity in Cyp1a1(+/-) but not in Cyp1a1(-/-) mice; TCDD pretreatment increases further the CYP1A1 in Cyp1a1(+/-) but not Cyp1a1(-/-) mice. Although a single 500 mg/kg dose of BaP was toxic to Cyp1a1(+/-) mice (serum liver enzyme elevated about 2-fold above control levels at 48 h), Cyp1a1(-/-) mice displayed no hepatotoxicity. Unexpectedly, we found 4-fold higher BaP-DNA adduct levels in Cyp1a1(-/-) than in Cyp1a1(+/-) mice; TCDD pretreatment lowered the levels of BaP-DNA adducts in both genotypes, suggesting the involvement of other TCDD-inducible detoxification enzymes. BaP was cleared from the blood much faster in Cyp1a1(+/-) than Cyp1a1(-/-) mice. Our results suggest that absence of the CYP1A1 enzyme protects the intact animal from BaP-mediated liver toxicity and death, by decreasing the formation of large amounts of toxic metabolites, whereas much slower metabolic clearance of BaP in Cyp1a1(-/-) mice leads to greater formation of BaP-DNA adducts.

Targeted knockout of Cyp1a1 gene does not alter hepatic constitutive expression of other genes in the mouse [Ah] battery.

Using the Cre-lox system, we have generated a cytochrome P450 1A1 Cyp1a1(-/-) knockout mouse by deletion of the translated portions of the Cyp1a1 gene. These mice are viable and demonstrate no obvious phenotype, compared with wild-type littermates. As a first step toward characterizing genes that might be expected to compensate for loss of CYP1A1, constitutive expression of [Ah] gene battery members was examined. In a cultured hepatoma CYP1A1 metabolism-deficient mutant line that does not express Cyp1a2, we have previously shown that constitutive transcriptional up-regulation of other [Ah] gene battery members occurs; these results are consistent with the elevation of a putative endogenous ligand (EL) for the Ah receptor that is a substrate for CYP1A1. The [Ah] battery includes Cyp1a2, NAD(P)H:quinone oxidoreductase (Nqo1), and three other Phase II genes. Examining mRNA, protein, and enzyme activity, we demonstrate that the absence of CYP1A1 has no effect on the hepatic constitutive expression of Cyp1a2 or Nqo1. We postulate that CYP1A1 and CYP1A2 might have overlapping substrate specificity for metabolism of the EL, such that basal CYP1A2 in the liver can compensate for the loss of CYP1A1.